Biochem Final
Anomeric carbon
New stereocenter formed via cyclization of monosaccharide
Know what enzyme complex does nitrogen fixation
Nitrogenase Complex
Which enzyme catalyzes nitrogen fixation?
Nitrogenase ezyme complex mediates the reduction of N2 to NH3
ΔG>0
Non-spontaneous ENDERGONIC energy is REQUIRED
Pyrimidine catabolism summary
Nucleoside is formed first Then complete degradation via ring opening reactions
CO binding of heme
O III C I Fe2+
Amino acid biosynthesis - Main reactions
One-carbon-transfers Transaminations
Thyroxine
Only found in the thyroid gland
Cell disruption methods preserving protein conformation physical
Osmolytic destruction Repeated freezing an thawing Shearing forces Ultrasound
The conversion of pyruvate into acetyl CoA commits the carbon atoms to either of two principal fates. What are the fates?
Oxidation to CO2 by the citric acid cycle or incorporation into lipids.
14) Why are aminoacyl tRNA synthetases really the only enzyme that knows the genetic code?
- Because they use the anticodon bases and other nucleotides on the tRNA molecule to ensure that the correct amino acid gets on the correct tRNA and also because they're very specific.
2) What are the 3 important sites of a ribosome and what occurs in each one?
- E Site (Exit): Exit site where empty tRNA molecules leave the ribosome - P Site (Peptidyl): Site where the Methionine-tRNA initially bind - A Site (Aminoacyl): Site where the Aminoacyl-tRNA molecule designated by the second codon on the mRNA binds
7) Why are GC and AT pairs important?
- GC bonds via 3 hydrogen bonds while AT bonds with only 2
9) What holds the DNA double helix together?
- Hydrogen bonding and Van Der Waal interactions
7) What are Complementary (cDNA) libraries
- Library that contains only the expressed genes
3) What are Southern, Northern and Western blotting
- Northern: The Northern blot is a technique used in molecular biology to study gene expression by detection of RNA - Southern: The same thing but for DNA - Western: It's an adaptation of the Southern Blot, used to identify specific amino-acid sequences in proteins.
Citric acid cycle intermediates
- Pyruvate - Acetyl-CoA - Citrate - Isocitrate - α-ketoglutarate - Succinyl CoA - Succinate - Fumarate - Malate - Oxaloacetate
8) What are ribozymes?
- Self-splicing RNAs
1) What are restriction enzymes nucleases?
- They are enzymes that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites.
13) What are aminoacyl tRNA synthetases?
- They are enzymes that link specific amino acids to specific tRNA molecules
Also know some of the downstream products of cholesterol (like bile salts) know where they are synthesized and where they are stored
- bile salts (detergents that solubilize dietary lipids): synthesized in the liver, stored in the gall bladder. vitamin D active form= calcitriol
Citric acid cycle enzymes
- citrate synthase - aconitase - isocitrate dehydrogenase - α-ketoglutarate dehydrogenase - succinyl-CoA synthetase - fumarase - malate dehydrogenase
1) What are the four types of RNA?
- mRNA, rRNA, tRNA and small micro RNA's
18) When you replicate eukaryotic DNA, what are the three challenges of replication in eukaryotes that are different form prokaryotes? How are they Solved?
1 Eukaryotic genomes are 100-1000X larger than Prokaryotic genomes 2 Eukaryotes have multiple chromosomes 3 Eukaryotic chromosomes are linear, not circular - Multiple origins of replications solve the first 2 problems - The presence of telomeres solve the 3rd problem
ELISA signal detection types
1) enzyme conjugated 2) biotinylated
Calvin cycle subdivision
1. Fixation of CO2 2. Recovery of RIbulose-1,5-Bisphophate - Preparation - Reshuffling - Isomerization - Phosphorylation
Vitamin A participation in vision
11-cis-Retinal is isomerized to 11-trans-Retinal
Peptide bond absorption
205 nm
Know number of carbons in: squaline: lanosterol: and cholesterol:
30 carbons; 30 carbons, 27 carbons
Aspartic acid structure
4 Cs -> D
6) In what direction does DNA synthesis proceed? RNA synthesis proceed? In what direction is mRNA read by the ribosome?
5' - 3' Direction
Photosynthesis net equation
6 CO2 + 6 H2O -> C6H12O6 + 6 O2
What are farnesyl and geranyl molecues?
A 15 and 10 carbon intermediate in the formation of squaline.
1° structure
AA sequence
Ketone bodies
Acetone Acetoacetate ß-hydroxybutyric acid
Know which enzyme in FA synthesis is the control point enzyme
Acetyl CoA Carboxylase
Vitamin E
Active form is α-tocopherol Traps HOO and ROO radicals (antioxidants)
purine salvage - adenine
Adenine + PRPP -[adenine phosphoribosyl transferase]-> AMP
Active transport
Against concentration gradient under consumption of energy
Chiral amino acids
All, but glycine
Know about ribonucleotide reductase
Allosterically regulates the synthesis of deoxyribnucleotides using NADPH
Filtration
HEPA filters remove all MOs larger than 0,3 μm
Know what atoms lead to purine and pyrimidine
Amino Acids - glycine, glutamine, aspartate, and tetrahydrofolate
Pre-step of FA synthesis
Biotin, ATP needed [Acetyl-CoA carboxylase]
Histidine 3- and 1-letter code
His H
Know about the relative normal saturation level of the fatty acids on carbon one and carbon two, carbon three is where the phosphate group is
C1 = Saturated C2 = Unsaturated
Hatch-Slack Pathway/C4-pathway
CO2 is fixed by Phophoenolpyruvate -> Oxaloacetate is reduced (NADPH -> NADP+) -> Malate gives CO2 into Calvin cycle (NADP+ -> NADPH) -> Pyruvate is phosphorylated twice (ATP->AMP) to phosphoenolpyruvate again
Glycogen phosphorylase
Catalyzes phosphorolysis of glycogen Glycogen + Pi -[glycogen phosphorylase]-> Remainder + G1P
Uncommon amino acids
Hydroxyproline Hydroxylysine Thyroxine Selenocysteine
IfSG
Causes of infectious disease
What is the actual second messenger in PIP2 mediated signal transduction
IP3 - induces Ca2+ release from intracellular reservoirs in the ER
Catabolism of unsaturated fatty acids
ISOMERASE induces cis-trans-isomerization Oxidation gives less energy
Cellulose
Cellubiose subunits ß-1,4 2x glucose
ΔG
Change in free energy ΔG = ΔH - TΔS
Vitamin D synthesis
Cholesterol via enzyme to 7-dehydrocholesterol Ultraviolet light breaks bond between C9 and C10 -> Vitamin D3 (Cholecalciferol) INACTIVATION: Made water soluble by 2 hydroxylations at C25 and C1 enzymatically
Prosthetic groups
Co-factors that are attached to an enzymes COVALENTLY
Staining methods for SDS-PAGE
Coomassie Silver (more sensitive) Fluorescent dyes
Cystein 3- and 1-letter code
Cys C
Checking for peroxidation
Determination of iodide ions
DAG
Diacylglycerol
How to identify if NAD+/NADH is reduced or oxidized
Different absorption maxima NAD+ 260 nm NADH 340 nm
Isoleucine 3- and 1-letter code
Ile I
Greek key
Motif from β-meanders
PhastGel IEF
Mutagenic -> gloves
β-meander
Domain
Role of glutamine and glutamate in amino acid reactions
Donors of amino groups
3° structure
Due to non-covalent and covalent interactions
Ketose
E.g. fructose
ELISA Non-competitive immuno assay
Excess of antibodies -> antibodies are labelled The more tested antigen binds the stronger signal
Thalassaemia
Excess of α-chains leads to precipitation in form of inclusion bodies which impair the functionality of RBCs α-globin cluster: chromosome 16 β-globin cluster: chromosome 11
Mechanism of oxidative phosphorylation
NADH donates 2 e- into electro transport chain While membrane proteins (Complex I, CoQ, Complex III, Cyt c, complex IV) are reduced and oxidized they take h+ from matrix release it into the intermembrane space This establishes a proton gradient between the mitochondrial matrix and intermembrane space Finally the 2 e- form H2O with 1/2 O2 from the mitochondrial matrix This further reduces [H+] in the mitochondrial matrix The proton gradient is used by F1F0 ATP synthase to phosphorylate ADP to ATP
Glucagon
Induces glycogen breakdown (increases blood sugar) Released by α-cells in islets of Langerhans in the pancreas
IP3
Inositol 1,4,5-triphophate
Interactions in H2O (4)
Ionic bonds i(+)i(-) Salt bridge e.g. in between amino acid side chains due to opposite charge Ion-dipole interactions Ions of buffer replace ionic bonds (like in IEC) Dipole-dipole interactions Between two dipoles
Keto- and Glucogenic amino acids (4)
Isoleucine Phenylalanine Tryptophan Tyrosine
Negative reaction (change in energy)
Free overall energy decreases ("down-hill") -> reaction can occur spontaneously
Phosphoglucomutase
G1P -[phosphoglucomutase]-> G6P In glycogen catabolism
Glutamine 3- and 1-letter code
Gln Q
Glycolysis pathway
Glucose Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-bisphosphate (Dihydroxyacetone phosphate) Glyceraldehyde-3-phosphate 1,3-phosphoglycerate 3-phosphoglycerate 2- phosphoglycerate Phosphoenolpyruvate Pyruvate
Proteinaceous amino acids are usually... (configuration)
L Exceptions: D-Ala in peptidoglycan Neurotransmitters Used in antibiotics
Ketogenic amino acids (2)
Leucine Lysine
Disk excitation cascade
Light absorption converts 11-cis-retianl to all-trans-retinal which activates rhodopsin Rhodopsin catalyzes replacement of GDP by GTP on transducin - dissociates into Tα- and Tβγ-subunit Tα-GTP activates cGMP phosphodiesterase (PDE) by removing its inhibitory subunit PDE reduces [cGMP] until cation channels close -> membrane is hyperpolarized; signal is passed to brain
Problem with 2D DIGE
Limited gel reproducibility -> different groups of proteins are differentially stained and loaded on one SINGLE gel
Why does the lack of glucose 6-phosphatase activity in the brain and muscle make good physiological sense?
Glucose is important energy source for both tissues and is essentially the only energy source for the brain. consequently these tissues should never release glucose. Glucose release is prevented by the absence of glucose 6-phosphotase.
Transamination reaction
Glutamate or Glutamine generally acts as an amino group donor for an α-ketoacid pyridoxalphosphat acts as carrier for the amino group binding it and binding to the enzyme [pyridoxal-phosphat-dependent amino transferase] (Schiff's base) After the reaction the amino acid donor is an α-ketoacid itself
Hemoglobin in Sickle-Cell anemia
Glutamic acid (E) [acidic] 6 in the β-chains is replaced by Valine (V) [non-polar]
Glycine 3- and 1-letter code
Gly G
What are some important intermediates of the non-oxidative part of pentose phosphate?
Glyceraldehyde 3-phosphate, Xylose 5-phosphate, and Fructose 6-phosphate
Enzymes involved in glycogen breakdown
Glycogen phosphorylase Debranching enzyme
Control of glycogen metabolism
Glycogen phosphorylase is controlled as well by phosphorylation as by allosteric effectors Inhibited by ATP, G6P, glucose (sufficient energy) Activated by AMP (energy needed) Activated by phosphorylation
Enzymes involved in glycogen synthesis
Glycogenin Glycogen synthase Branching enzyme
Gangliosides
Glycolipids with complex carbohydrate moiety that contains more than 3 sugars One of them is Sialic acid
Overall pathways
Glycolysis has pyruvate as product Pyruvate either can be 1) further oxidized in CAC and oxidative phosphorylation or 2) anaerobically metabolized to lactate to get NAD+ back [Anaerobic Glycolysis] or 3) Anaerobically fermented to ethanol to get NAD+ back
What feeds into CAC
Glycolysis in form of Acetyl-CoA FA oxidation in form of Acetyl-CoA Amino acids can be converted to Pyruvate Acetyl-CoA α-ketoglutarate Succinyl-CoA Fumarate Malate
Methionine 3- and 1-letter code
Met M
Know about the steroid hormones and some of the other products of cholesterol biosynthesis
steroid hormones are important for secondary sex characteristics, embryonic differentiation, the maintenance and regulation of cycles (menstruation), regulation of glucose metabolism and inflammation, and the regulation of salt balance and blood pressure.
Hypoxanthine
uncommon base like guanine but without NH
βαβ
α-barrel is linker between 2 β-sheets Domain
Intermediates in glycolysis from which amino acids can be formed
- 3-PG (Serine family) - Phosphoenolpyruvate (Aromatic family) - Pyruvate (Pyruvate family)
6) What is Ricin and what does it do?
- A Ribosomal inhibiting protein that cleaves an adenine from the 28S rRNA molecule in eukaryotes. This prevents the binding of elongation factor, disrupting protein synthesis.
16) What are codons and anti-codons?
- A codon is a three-base sequence (three nitrogen bases in a row) on mRNA. It calls for a specific amino acid to be brought to the growing polypeptide. - An anticodon is a three-base sequence on tRNA. It matches the codon. That's how the right amino acid is put onto the polypeptide next. The tRNA must fit its anticodon onto the mRNA codon like a jigsaw puzzle piece. Each tRNA can only bring one kind of amino acid.
10) What is a micro array?
- A microarray is a sequence of dots of DNA, protein, or tissue arranged on an array for easy simultaneous analysis. The most famous is the DNA microarray, which plays an integral role in gene expression profiling.
5) What are DNA vectors?
- A piece of DNA readily taken up and replicated by bacteria
7) How does the peptide bond formation occur?
- A region on the 23S rRNA molecule of the Large Subunit enables a peptide bond to form between the carboxyl terminus of the fMet and the free amino terminus of the aminoacyl-Trna
2) What is the Ames test? How was it formulated? What organism is used? What modifications make it more relevant to eukaryotes?
- A strain of Salmonella typhimurium that has a defect in its histidine biosynthetic pathway (and must grow on media supplemented with the amino acid histidine) is exposed to the potential carcinogen. The number of revertants (above background levels) that grow on media that does not contain histidine, is a measure of the strength of mutagenic agent.
12) What is the start codon?
- AUG is the "start" codon and codes for the amino acid Methionine
6) What is alternative splicing?
- Alternative splicing is a mechanism by which multiple protein products can be produced from a single primary transcript by alternating how the transcript is spliced
2) What is a helicase?
- An enzyme that binds and unwinds DNA
What is ligase?
- An enzyme that seals the "nicks" by making phosphodiester bonds between Okazaki fragments.
14) Why do eukaryotes have to compact their DNA? How do they use it? How do they do it?
- Because the linear length of DNA is nearly 1,000 times the length of a single cell. They use supercoiling to compact the DNA. Eukaryotes wind around proteins like wire around a bale of hay.
11) What is Watson-Crick Base Pairing? Which DNA did they use A,B or Z?
- Cytosine binds with Guanine via 3 hydrogen bonds Adenine binds with Thymine via 2 hydrogen bonds They used B DNA
6) Why is DNA synthesis semi-discontinuous?
- DNA replication is called semi-discontinuous because one strand is synthesized continuously (the leading strand) and the other strand is synthesized discontinuously in small fragments (the lagging strand).
What are the forces that stabilize DNA structure?
- DNA structure is stabilized by the hydrogen bonding between base pairs and by van der Waals interactions that result from the stacking of bases
3) Why do organisms use Thymine instead of Uracil in DNA?
- Despite uracil's tendency to pair with adenine, it can also pair with any other base, including itself. By adding a methyl group (which is hydrophobic) and turning it into thymine, its position is reorganized in the double-helix, not allowing those wrong pairings to happen
8) What is Sanger or Dideoxy sequencing
- Dideoxy sequencing (also called chain- termination or Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying lengths, stopping DNA replication at one of the four bases and then determining the resulting fragment lengths.
17) What are stem-loop structures?
- Double stranded regions within a single RNA molecule, involved with regulation. RNA will form double-stranded regions within a single molecule that are called secondary structural elements. These secondary structural elements are termed stem-loop structures
Hormonal amplification
- Each time epinephrine or glucagon binds to its specific receptor, it activates a number of stimulatory G proteins - Each activated G protein, in turn, stimulates adenylate cyclase a number of times which increases the production of cAMP
19) What are telomeres?
- Ends of chromosomes which keep the linear chromosome from shortening each time they are replicated
2) What is the genetic structure of eukaryotic genes (introns and exons)? What extra burden do these impose on transcription?
- Exons are regions of the gene that code for the amino acids that make up the protein that is produced in translation (they are expressed). - Introns are intervening sequences in the genes that although they are part of the primary transcript, are spliced out during processing and do not code for amino acids in the protein product.
10) What are post-transcriptional modifications of ribosomal RNA and tRNA in prokaryotes?
- For rRNA and tRNA molecules post-transcriptional modifications are usually required. A primary transcript may contain more than one gene product which all need to be cleaved out of larger molecules. Ribonucleases are responsible for these cleavages
1) What did Griffith have to do with the concept of transformation?
- Griffith used Streptococcus pneumoniae in mice and inserted a dead (heated) III-S smooth (virulent) and II-R rough (nonvirulent) strain into the mouse and the mouse died. When the II-R strain was put in the mouse by itself, the mouse lived; and vice versa for the III-S strain. He concluded that the II-R strain transformed into the III-S using DNA as a "transforming factor."
16) What proteins make up the octamer of the nucleosome? What does H1 do?
- Histone proteins form an octamer with a + charge. The (-) phosphodiester backbone of the linear DNA wraps around the octamer. - Histone protein H1 then helps the DNA-protein octamers associate. Other proteins then help the further condensation of DNA to form chromatin
4) What are some examples of hormones that affect transcription? (estrogen, estradiol, thyroid)
- Hormones (like estradiol) are hydrophobic and can diffuse across the cell membrane and attach to receptor proteins inside the cytoplasm or nucleoplasm. - When estrogen is not bound to the receptor protein, it is inactive and transcription is not initiated while. - In the case of thyroid hormone when their receptors are bound transcription is repressed because the proteins can bind corepressor proteins that inhibit transcription.
15) What are the mechanisms that ensure the correct amino acid is put on the correct tRNA molecule?
- In addition to the specificity of incorporation due to specific active site interactions with the incoming amino acid, the aminoacyl-tRNA synthetase also has an editing site separate from the active site. - The CCA-amino acid "arm" can swing from the active site to the editing site so that it can check to see if the correct amino acid is attached. If it is not, then the bond is hydrolyzed and the enzyme can try to attach the correct amino acid again. - Amino acids larger than the correct amino acid won't fit into the active site, and only smaller amino acids will fit into the editing site. This is an elegant economy of function. - Aminoacyl-tRNA synthetases also use the anticodon bases and other nucleotides on the tRNA molecule ( to ensure that the correct amino acid gets on the correct tRNA.
13) What are inducible and repressible operons? What is Catabolite Activator Protein?
- Inducible Operons - transcription is normally shut off, and can be turned on under certain circumstances - Repressible Operons - When an inducer binds to a repressor, it changes the conformation so that it can no longer bind to the operator - CAP - Catabolite Activator Protein - bind cAMP and changes its conformation so that it can bind to promoter regions and recruit RNA polymerase
1) What are the 3 steps of translation?
- Initiation: The initiator codon (AUG) is lined up correctly by using a region upstream of AUG start codon to position it (Shine-Dalgargno Sequence). Once the mRNA is lined up correctly with the 30S subunit, the Met-tRNA in the P site of the ribosome. - Elongation: the A site is unoccupied and has the second codon on the mRNA molecule in it. Elongation factor Tu brings in the amionacyl tRNA-synthetase whose anticodon will correctly complementary base pair with the second codon. - Termination: When a stop codon is reached, Release Factor interacts with the ribosome to hydrolyze the bond in the final aminoacyl tRNA molecule.
13) What enzymes help prokaryotes supercoil their DNA? Why do they even have to supercoil their DNA?
- It is done in order to compact the DNA - supercoiling it is achieved by enzymatic activity by topoisomerases. Prokaryotes anchor their DNA and wind like a rubber band
7) What is the function of the 7-methyl guanosine cap? polyA tail?
- It stabilizes mRNAs by protecting their 5' ends from phosphatases and nucleases. Caps enhance the translation of mRNA by eukaryotic protein-synthesizing systems. - The presence of the polyA tail enhances the translation efficiency of the mRNA as well as increases its stability.
6) What is a genomic library?
- Library that contains the entire Genome
4) For Eukaryotes, know what eukaryotic ribosomes small subunits use to position the start codon in the P site in eukaryotic RNA
- Met-tRNA is used instead of FMet-tRNA for initiation. The small subunits use the 5' cap on the mRNA molecule as a starting point and just moves down until it finds the first AUG codon and starts there.
1) What do the terms mismatch repair, base excision repair, and nucleotide excision repair mean?
- Mismatch Repair - the template strand is methylated, and the newly synthesized strand has not been methylated. An exonuclease excises out a region around and including the error. - Base Excision Repair - the nucleotide base is clipped from the DNA backbone by a glycosylase. AP endonuclease nicks the backbone. DNA pol I inserts a replacement nucleotide and ligase seals the nick - Nucleotide Excision Repair - UvrABC enzyme system cuts out a region of DNA including the dimer and DNA pol I fills the gap and ligase seals it.
11) What is the tri-nucleotide sequence and where all tRNA's ended?
- NA Polymerase III transcribes transfer RNAs (tRNAs). The 5' end is trimmed by RNase P, the 3' end is trimmed and the trinucleotide sequence CCA is added (if necessary), and ribose sugars and nucleotide bases are extensively chemically modified. - Many eukaryotic tRNAs also undergo splicing to remove internal sequences. - Nucleotides may be added to the 3' ends of some RNA molecules. ALL tRNA molecules end in the sequence CCA, which is added to tRNAs that do not already have it. - In tRNAs a variety of chemical modifications occur, including the conversion of uracil to pseudo uracil, or thymine
2) Be able to tell if a DNA sequence is palindromic or not
- Palendromic means that the DNA sequence reads the same in either direction (right to left or left to right). Example: racecar, mom.
3) How many RNA polymerases are there in prokaryotes and eukaryotes?
- Prokaryotes - 1 - Eukaryotes - 3
18) What are the differences in the sizes of large and small subunits in prokaryotes vs eukaryotes?
- Prokaryotes: 70S ribosome made of: 50S Large Subunit (consisting of the 23S rRNA, the 5s rRNA, and 34 proteins) and a 30s Small Subunit (consisiting of the 16S rRNA and 21 proteins). - Eukaryotes: 80S ribosome made of: 60S large subunit (5S rRNA and 28S rRNA, 5.8S rRNA and 46 proteins) 40S small subunit (18S rRNA and 33 proteins)
3) In prokaryotic replication: where is it done, what enzymes are involved, and how is it done? What is primase?
- Prokaryotic replication takes place at an origin or replication (eg. in E. coli - OriC) DnaB (an ATP-hydrolyzing helicase) unwinds DNA strands and single-strand-binding proteins stabilize the single stranded regions - Primase synthesizes a short region of RNA complementary to the DNA strand to provide a free 3' OH group for DNA synthesis. Primers are removed by 5' 3' exonuclease prior to the completion of replication
What are promoters, sigma factors, terminators and Rho proteins?
- Promoters - The regions that occur in the 5' region before genes and are recognized by the sigma factor of RNA polymerase in prokaryotes. - Termination of RNA synthesis may also occur due to the interaction of Rho proteins, which bind to the growing RNA chain and finds regions rich in Cytosine and poor in Guanonine. Rho moves down the chain and finds the RNA pol causing the RNA to dissociate from the DNA template.
12) What are major and minor grooves in DNA structure? Where do proteins usually interact? Why?
- Proteins usually interact with DNA through the major groove, since they have greater access and the opportunity to hydrogen bond with the nitrogenous bases. This enables the proteins to find and interact with specific sequences in the DNA double helix.
4) What classes of genes do eukaryotic RNA polymerases I, II, and III synthesize?
- RNA Polymerase I transcribes ribosomal RNAs - RNA Polymerase II transcribes messenger RNAs and small nuclear RNAs - RNA Polymerase III transcribes the 5S ribosomal RNA and tRNAs.
7) What subunits make up the core and holoenzyme of RNA polymerase?
- RNA polymerase in prokaryotes consists of 5 subunits (α2ββ'σ). - The sigma subunit (σ) finds the promoter region (where transcription begins) and the α2ββ' subunits are called the core enzyme. - The catalytic site is at the interface of the β and β' subunits.
4) What is homologous and non-homologous recombination?
- Recombination - Two "parent" DNA molecules exchange sections of DNA to form two "daughter" molecules. - Homologous - (between regions that are somewhat similar in sequence) - Non-homologous - (regions share no similarity)
4) What does topoisomerase do?
- Relax supercoiled DNA so that helicases can unwind the DNA strands - Type 1 topoisomerases relax supercoiling - Type 2 topoisomerases (gyrase) add supercoils to DNA and require ATP hydrolysis
4) What is reverse transcriptase?
- Reverse Transcriptase (RT) is an anzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription.
10) What are the 4 things you need for translation to occur?
- Ribosome - Messenger RNA (mRNA) molecule - Aminoacyl-tRNA molecule - Enenrgy
17) What are ribosomes?
- Ribosomes are organelles that consist of RNA and proteins. Ribosomes are composed of a large subunit and a small subunit. Ribosomal subunits are synthesized by the nucleolus. These two subunits join together when the ribosome attaches to messenger RNA (mRNA) during protein synthesis. Ribosomes along with another RNA molecule, transfer RNA (tRNA), help to translate the protein-coding genes in mRNA into proteins.
12) What antibiotics interfere with transcription?
- Rifampicin binds to a pocket in RNA Polymerase, blocking chain elongation - Actinomycetes binds to DNA by intercalating between the base pairs of DNA, making it a poor template for transcript
What are Okazaki fragments?
- Short segments of DNA that are synthesized on the lagging strand of DNA. These fragments are subsequently joined by DNA ligase, forming a continuous segment of DNA
5) What is splicing out of introns? How does it happen? Associated factors?
- Splicing - the excision of introns from the primary mRNA transcript. - Takes place at the ribonucleoprotein complex called the spliceosome consisting of small nuclear RNAs (snRNA) and specific proteins called small nuclear ribonucleoproteins (snRNP) - U1 snRNP binds at the 5' splice site, followed by U2 snRNP binding at the branch site. - Binding of U4-U5-U6 tri-snRNP completes spliceosome formation.
Hormone types
- Steroids (estrogens and androgens) [nuclear receptors] - Polypeptides (insulin and glucagon) - Amino acid derivatives (epinephrine)
3) What is TATA Binding Protein?
- Tata Binding Protein starts the process of initiation by binding to the TATA box. It is a saddle shaped protein and induces conformational changes in the DNA when it bind, unwinding it and opening up the minor groove
3) What is the Shine-Delgarno sequence in prokaryotes?
- The Initiator codon is lined up correctly by using a region upstream of the AUG start codon to position it. This region on the mRNA is called the Shine-Dalgarno Sequence and is complementary to a region on the 3' end of the 16S SSU rRNA molecule in the 30S fragment of the ribosome.
2) What are the prokaryotic replication structures?
- The Replisome is a complex of proteins that includes two DNA pol III enzymes, Primase, Helicase, DNA polymerase I, and DNA ligase
7) What is the clamp for DNA polymerase III? What does clamp-loading protein do?
- The b2 subunit of DNA Polymerase III forms a DNA clamp on the template and keeps DNA Polymerase III from falling off the template. A clamp loader protein helps the clamp get on the template
1) What is RNA polymerase II in eukaryotes? What are some special things about it?
- The eukaryotic RNA Polymerase II contains a unique Carboxyl-Terminal Domain that enables it to be regulated via phosphorylation of Serine residues.
14) What is the lac operon and how does it work?
- The lac operon consists of genes for the β-Galactosidase, a lactose permease, and a transacetylase. "is an operon required for the transport and metabolism of lactose in E coli and some other enteric bacteria" - One of the genes encoded by the lac operon produces the enzyme β -Galactosidase, which hydrolyzes the β 14 bond in the disaccharide lactose (galactose β 14 glucose).
9) Know about PCR -
- The polymerase chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold. As the name suggests, the process creates a chain of many pieces, in this case the pieces are nucleotides, and the chain is a strand of DNA. - General Outline: 1. Double Helix containing the targer sequence and flanking regions (primers) is heated to 95oC to denature the double helix and form single strands. 2. Mixture is cooled (55oC) to allow the primers to anneal (bond) to template DNA. 3. Mixture is heated to 72oC, allowing the polymerase to synthesize DNA. - The 3 steps are repeated 20-30 times to generate large amounts of target DNA.
5) RNA polymerase makes errors much more often than DNA polymerase. Why is that not a problem?
- This is acceptable since the mistakes are not passed on to progeny - Due to the degeneracy of the genetic code and the fact that often most amino acid changes do not have a measurable effect on protein function, - Finally due to the fact that mRNA molecules can be used multiple times in translation
1) Review the Messelson and Stahl experiment that is in the book
- This is called semiconservative replication, and was determined by a series of experiments conducted by Meselson and Stahl where they labeled parental strands of DNA with the "heavy" isotope of Nitrogen (N15). They then switched the cells to the light isotope (N14) for one generation. They detected one band, which meant that replication could have been either semiconservative or random (also called dispersive), but eliminated the possibility of replication being conservative. By continuing the experiment they were able to determine that replication was semiconservative and not random.
2) What are the similarities between DNA polymerase and RNA polymerase? Where are the catalytic sites? What divalent cation is important in catalytic mechanism?
- Transcription is the process by which the information stored in DNA is transferred to a soluble RNA molecule. - The enzyme that catalyzes the polymerization of ribonucleotide triphosphates into a polymeric RNA molecule is called RNA Polymerase. - RNA polymerase is a multimeric enzyme that requires a template molecule that is usually DNA (but can be RNA in some cases), ribonucleotide triphosphates, and a divalent cation (Mg2+) in its catalytic mechanism. - In RNA, the thymine used in DNA is replaced by uracil. - The synthesis of RNA is like that of DNA, in that when each nucleotide monophosphate is incorporated into the growing polymer, pyrophosphate (PPi) is released. - The direction of synthesis is 5' 3' and requires a free 3' OH group for the continuation of the chain. RNA polymerase does NOT require a primer. - Genes are segments of DNA that encode RNA transcriptional products.
9) How is mRNA processed?
- mRNA molecules are translated in the cytoplasm. There is a spatial and temporal disconnect between transcription and translation in eukaryotes that is not seen in prokaryotes. This disconnect enable eukaryotes to regulate transcription and translation much more finely that prokaryotes are able to.
11) What does it mean when the code is said to be degenerate?
- multiple codons code for the same amino acid
Selecting a buffer
- suitable ionic strength - pKa = pH - non-biological nature - no interference - suitable solubility
20) What is telomerase?
- the enzyme that synthesizes telomeres on the 3' ends of chromosomes, enabling primers to be made for replication without shortening the chromosomes each time they are replicated. - Adds nucleotides to the leading strand so that the lagging strand will always maintain its approximate length. Contains an RNA molecule that acts as a template for extending the leading strand.
Intermediates in citric acid cycle from which amino acids can be formed
- α-ketoglutarate (Glutamate family) - Oxaloacetate (Aspartate family)
Pentose Phosphate Pathway - 2 most important products
-NADPH, Ribose 5-Phosphate
Ethers general structure characteristic functional group
-O- midst molecule (ether group) e.g. CH3-O-CH3
Know how regulation of pentose phosphate occurs, and how it centers on NADP+ concentration
-Regulation shunts Glucose 6-phosphate to where it is most needed (PPP or glycolytic pathway) - Reaction catalyzed by G6P Dehydrogenase is essentially irreversible in the PPP -Low concentrations of NADP+ = low activity of G6P Dehydrogenase and therefore more G6P to glycolytic pathway
H2O covalent bond length van der Waals radius of H and O
0.095 nm H-O 0.14 nm (O) 0.12 nm (H)
Classification codes 1 - 3
1 oxidoreductases 2 transferases 3 hydrolases
Colorimetric assays ordered by sensitivity
1) Bradford assay 2) BCA assay 3) Lowry assay 4) Biuret assay
Edman method (4)
1) COUPLING - Phenylisothiocyanate 2) CLEAVAGE 3) CONVERSION 4) AA of interest is bound to PTH derivative to be analyzed by chromatographic methods
Determination of protein primary sequence Steps (4)
1) Identify relative amounts of AAs hydrolization of peptide bonds (heat + low pH) Separate and identify AAs by HPLC -> to know which cleavage methods can. be applied in step 3 2) Identify N- and C-terminal amino acids By specific reagents 3) Cleave protein by different proteases (e.g. trypsin, chymotrypsin) or chemical reagents (cyanogen bromide) -> OVERLAP is NEEDED to keep track of order of fragments 4) Determine sequence of smaller peptide fragments by EDMAN METHOD
Production of monoclonal antibodies
1) Inject AG 2) Kill, isolate B cells from spleen 3) Fuse with myeloma(cancer-)cell line (immortalization) 4) AG-binding assay 5) Expand & purify reactive ABs -> can be kept for decades
Unique reactions of the glyoxylate cycle
1) Isocitrate -> Succinate + Glyoxylate [ISOCITRATE LYASE] 2) Glyoxylate + acetyl-CoA -> Malate [MALATE SYNTHASE]
NADPH generation (2 ways)
1) MAINLY PENTOSE PHOSPHATE PATHWAY 2) Citrate crosses mitochondrial membrane where it is split into acetyl-CoA (goes into FA synthesis) and oxaloacetate Oxaloacetate -[malate dehydrogenase]-> malate -[malic enzyme]-> pyruvate This generates NADPH and recycles Pyruvate
Light reaction players
1) PS II 2) PQ - plastoquinone 3) Cyt b6 - f 4) PC - plastocyanine 5) PS I 6) enzyme NADP+ -> NADPH 7) ATP syntase
Gradient for ATP production establishment
1) Splitting of H2O releases H+ into thylakoid 2) Transport of H+ into thylakoid by Cyt b6-f complex 3) NADP+ reduction to NADPH takes away H+ from stroma
Steps of fatty acid catabolism
1) activation -> RCO-S-CoA 2) transport into mitochondria (carnitine and carnitine acyltransferase) 3) ß-oxidation
Purine catabolism
1) deribosylated and dephosphorylated (free base) 2) converted to xanthine [GMP is converted directly to xanthine; AMP is converted to hypoxanthine and then to xanthine] Does not yield any energy despite purine anabolism requiring a lot of energy Xanthine is then further catabolized
Van der Waals forces
1) dipole-dipole interactions 2) dipole induced-dipole interactions e.g.: O=O is distorted by H2O 3) dipole induced-dipole induced interactions -> bonpolar molecules have some attraction for one another
Affinity chromatography elution methods and problems with these
1) excess (may be expensive) 2) pH or salt c change (may damage protein of interest)
Production of polyclonal antibodies
1) inject AG & immunostimulant into donor animal 2) boost by 2nd AG injection 3) take blood sample 4) purify reactive ABs -> may still react if target protein undergoes minor changes
Emzyme-Substrate binding models
1) lock and key model: complementary fit is present all the time 2) induced fit model: complementary fit is triggered by E-S binding
Amino acid catabolism steps
1) removal of amino group by transamination - transfer to form glutamate Glutamate then either is used for biosynthesis or excreted as nitrogenous product 2) breakdown of carbon skeleton 2 pathways: a) for glucogenic amino acids b) for ketogenic amino acids
Fatty acid nomenclature e.g. 16:1-Δ^9
16 carbon atoms 1-fold unsaturated (1 double bond) at carbon atom 9 counted from carboxylic acid site
Palmitate
16:0
Palmitic acid
16:0
Palmitoleic acid
16:1-Δ9
Stearic acid
18:0
Oleic acid
18:1-Δ9
Oleic acid
18:1-∆9
Risk groups 1 - 4
1: no/low individual nor community risk 2: moderate individual risk, low community risk 3: high individual risk, low community risk 4: high individual and community risk
Pentose-phosphate pathway reactions
2 oxidation steps Brings ribulose-5-phosphate (for NA synthesis) and NADPH Multiple reshuffling steps from which 3, 4, 5, 6 and 7 C monosaccharide-phosphates are formed Among these: Glycolytic intermediates Fructose-6-P Glyceraldehyde-3-P
αα unit
2 α-barrels Domain
BPG
2,3-Bisphophoglycerate Allosteric effector of Hb Triggers conformational change if bound Makes sure fetal HbF gets oxygen
Arachidonic acid & Prostaglandins
20 carbons Prostaglandins derived from arachidonic acid Multiple forms and functions
Reference measurement for proteins
260 nm because nucleic acids absorb at 280 nm, too
Cholesterol structure
27 Cs
Trp absorption
280 nm
Detection of protein phosphorylation
2D gel electrophoresis MW stays the same but pI shifts Uncharged AA becomes negatively charged
Maltose
2x D-glucose α-1,4
Cellubiose
2x D-glucose ß-1,4 linked
Collagen triple helix
3 PP chains wrapped around each other Each of three polypeptide chain is a helix itself (not an α-helix) Highly repetitive sequence: -X-Pro-Gly- -X-Hyp-Gly- Hyp = Hydroxyproline Hydrogen bonds between HYP and HYL With age: covalent bonds between HYL and LYS
E.coli AP
3 metal binding sites M1 M2 M3 Inhibition is tested by removing Zn ions By 8-hydroxychinoline
Cholesterol biosynthesis: know what the rate limiting enzyme is (complete name) and how its transcription is regulated
3-Hydroxy-3-MethylGlutaryl CoA Reductase. Regulated by Sterol Regulatory Element Binding Protein
Glutamic acid structure
4 Cs -> E
Products from Citric acid cycle
4 NADH + H+ 3 CO2 FADH2 ATP
Classification codes 4 - 6
4 lyases 5 isomerases 6 ligases
*Taken care of the nitrogen of amino acids, now we have to deal with the carbon skeleton. There are two major pathways or cycles into which they flow, know what they are:
: Glycolytic Pathway, or the Citric Acid Cycle
Electronegativity
A measure of the ability of an atom in a chemical compound to attract electrons O and N are more electronegative than carbon and hydrogen -> results in polar bonds
Co-factor
A nonprotein molecule or ion that is required for the proper functioning of an enzyme. May be a metal or an organic molecule Not every co-factor is a coenzyme but every coenzyme is a co-factor
A vs B vs Z-DNA
A-DNA and B-DNA both right-handed Z-DNA is left-handed
What is the effect of each of the following inhibitors on electron tranport and ATP formation by the respiratory chain? A. Azide. B. Atractyloside. C. Rotenone. D. DNP. E. Carbon monoxide. F. Antimycin A.
A. Azide blocks electron transport and prtoton pumping at Complex IV. B. Atractyloside blocks electron transport and ATP synthesis by inhibiting the exchange of ATP and ADP across the inner mitochondrial membrane. C. Rotenone blocks electron transport and proton pumping at Complex I. D. DNP blocks ATP synthesis without inhibiting electron transport by dissipating the proton gradient. E. Carbon monoxide blocks electron transport and proton pum[omg at Complex IV. F. Antimycin A blocks electron tranport and proton pumping at Complex III.
Cross reactivity
AB binds to another protein than supposed
What is needed in biotin assisted reactions?
ATP, Mg2+
Light reaction products
ATP, NAPH and O2
Fatty acid synthesis: Know ATP citrase liase, acetyl CoA carboxylase (know substrates & products of the enzymes)
ATP-Citrase: Converts Citrate, ATP, CoA, and H2O -> Acetyl CoA, ADP, Pi, and Oxaloacetate Acetyl CoA Carboxylase: Acetyl CoA, ATP, HCO3, -> Malonyl CoA, ADP, Pi, and H+
Fatty acid biosynthesis
Acetyl-ACP and Malonyl-ACP Per cycle, 2 carbon atoms are added in form of Malonyl-ACP Malonyl is then completely saturated H3C-CH2- Finally H2O is added and ACP-SH is released Palmitate: Saturated FA; 16 carbon atoms
Reducing sugar
Acting as reducing agent Oxidation of cyclic hemiacetal gives LACTONE Happens at anomeric carbon If anomeric carbon in bonding (glycosidic linkage) Tollens test is negative If anomeric carbon is not in bonding and free Tollens test is positive (anomeric carbon is oxidized)
ß-oxidation intermediates and enzymes
Acyl-CoA [acyl-CoA dehydrogenase] Trans-∆2-enoyl-CoA [enoyl-CoA hydratase] ß-hydroxyacyl-CoA [ß-hydroxyacyl-CoA dehydrogenase] ß-ketoacyl-CoA [thiolase] Acety-CoA + shortened acyl-CoA
dUTP can be converted to dTTP
Addition of a methyl group by thymidylate synthase Tetrahydrofolate serves as one carbon carrier This process is target for cancer therapy: Rapidly proliferating cells need deoxynucleotides for DNA synthesis - this is inhibited
Glycogen synthase
Adds UDPG to growing glycogen chain catalyzing α-1,4 linkages
Alanine 3- and 1-letter code
Ala A
Ultimate Degradation Product Amino Acids Pyruvate: Oxaloacetate: α-ketoglutarate Succinyl CoA Acetyl CoA Fumarate
Alanine, cysteine, glycine, serine, threonine, and tryptophan Aspartate and asparagine Arginine, glutamate, glutamine, histidine, and proline Threonine, methionine, valine, and isoleucine Leucine, isoleucine, and valine Aspartate, phenylalanine, and tyrosine
It can be argued that if life were to exist elsewhere in the universe it would require some process like photosynthesis. Why is this argument reasonable? If the starship enterprise were to land on a distant planet and find no measurable oxygen in the atmosphere, could the crew conclude that photosynthesis is not taking place?
All ecosystems require an energy source from outside the system, because the chemical energy sources will ultimately be limited. The photosynthetic conversion of sunlight is one example of such a process. Other chemicals besides water could be donated electrons and protons.
What is the evidence that modern mitochondria arose from a single endosymbiotic event?
All mitochondria contain at least 2% of the same DNA. This suggests a single evolutionary event.
Catabolism of odd numbered fatty acids
Also ß-oxidation Last beta oxidation cycle gives propionyl-CoA
Epinephrine
Amino acid derived hormone Molecular structure is closely related to Tyrosine Acts on muscle to raise glucose levels
Amines general structure characteristic functional group
Amino group NH2 e.g. CH3-NH2
Salting-out
Ammonium sulfate is added Ammonium sulfate makes proteins insoluble taking away water by interacting with it -> Exploited for protein purification Best soluble proteins elute last
Starch
Amylose and amylopectin Amylopectin α-1,4 and α-1,6 (24-30 SUs) Amylose α-1,4 (4000 SUs) Glucose subunits
Know what ketone bodies are, why we use them, what tissues use them, why we make them,
An alternative fate of the Acetyl CoA formed in β oxidation (formed in the liver). -Water soluble, can be a major energy source for the brain and heart in starvation conditions. -Preserves glucose for other tissues' needs
Hormone
An intracellular messenger produced from glands of the endocrine system Transported via bloodstream
2° structure of DNA
Antiparallel double helix
Arginine 3- and 1-letter code
Arg R
Reactions in cytoplasm
Argininosuccinate synthetase: Citrulline + aspartate + ATP -> argininosuccinate + AMP + PPi Argininosuccinase: Argininosuccinate -> arginine + fumarate Arginase: Arginine -> ornithine + urea
C3 plants are most common in higher latitudes and become less common at latitudes near the equator. The reverse is true of C4 plants. How might global warming affect this distribution?
As global warming progresses C4 plants will invade the higher latutudes where as C3 plants will retreat to cooler regions.
Asparagine 3- and 1-letter code
Asn N
Aspartic acid 3- and 1-letter code
Asp D
Where pyrimidine biosynthesis is regulated (rate limiting step
Aspartate transcarbamoylase
Acidic amino acids (4)
Aspartic acid Glutamic acid (Cysteine) (Tyrosine)
Essential amino acids have longer and more complex pathways. Can make 3 amino acids by a simple transamination reaction.
Aspartic acid, glutamic acid, and alanine
Phosphorylation of enzymes mechanism
At Ser/Thr/Tyr's OH group By protein kinase UNDER ATP CONSUMPTION Removed by phosphatase PHOSPHATE ESTER is formed at OH group
Differentiate between autotrophs and heterotrophs.
Autotrophs can use the energy of sunlight, CO2 and water to synthesize carbohydrates, which can subsequently be used for catabolic or anabolic purposes. Heterotrophs require chemical fuels and are thus ultimately dependent on auttrophs.
10) What are A, B, and Z DNA? Which is Most Important?
B - The DNA double helix studied by Watson and Crick is called "B" DNA (Figure 32.17) and is the most common structure. It is a right-handed double helix. A - DNA can also adopt the "A" conformation, which is a right-handed double helix, but the basepairs are tilted. "A" DNA is the form dehydrated DNA adopts, but is also that formed by DNA-RNA hybrids (important in regulation) and by double-stranded RNA molecules. Z - DNA can adopt the "Z" conformation, which is a left-handed double helix. The biological import of "Z" DNA is still being determined.
Biosafety level vs security level
BSL: L Security level: S
Glyceraldehyde 3 phosphate dehydrogenase in chloroplasts uses NADPH to participate in the synthesis of glucose. In gluconeogensis in the cytoplasm the isozyme of dehydrogenase uses NADH. Why is the use of NADPH by the chloroplast advantageous?
Because NADPH is generated in the chloroplasts by the light reactions.
Fatty Acids- 6 carbons of the fatty acid produce 42 ATP 6 carbons of glucose produce 30-32 ATP -This is why 6 C of fatty acid is better storage material for energy than glucose -Why lipids > CHO for storage Where does the 42 and the 32 come from in ATP numbers?
Because of substrate level phosphorylation, there are 14 ATP produced for every 2 Carbons.
In an atmosphere devoid of CO2 but right in O2 the oxygenase acrivity of rubisco disappears. Why?
Because the carbamate forms only in the presence of CO2 this property would prevent rubisco from catalyzing the oxygenase reaction when CO2 is absent.
Know about carbonoilphophate synthesis #2, (perimidine) substrates/products
Bicarbonate + Ammonia + 2 ATP -> Carbamoyl phosphate (3 step reaction)
UNcompetitive inhibitors
Binding to ES to form ESI to prevent enzyme from reacting with substrate to form product
Carbon dioxide binding Hb
Binds to N terminus of each polypeptide chain as carbamate group releasing H+
Lowry assay
Biuret reaction catalyzes reduction of Folic reagent (blue colour) + higher sensitivity + SDS tolerant - not tolerant to reducing agents, ammonium sulfate - depends on AA composition (aromatic AAs! [TYR])
Phosphorylation of enzymes - activation or inactivation
Both possible
Glycogen
Branched polysaccharide composed from glucose subunits α-1,4 linked linear chains Branches are α-1,6 linked
**If you break down palmitoleoyl CoA, know how many ATP you get from palmitic acid and palmitoleic acid, also know how many CoA you need to completely breakdown palmatoil CoA with B oxidation.
Break down palmitate (16C FA), 106 ATP is produced. Need 7 Acetyl CoA???
Buffer systems of the human body
Buffer in cells: H2PO4-/HPO42- Buffer in blood: H2CO3/HCO3-
Pyrimidine biosynthesis is a linear pathway, what are the major steps along that pathway? So we can get to UTP CTP and TTP
Build a ring, make carbamoylaspartate and pi turn that into orotate which is then made into UMP
Conversion of ribonucleotides to deoxyribonucleotides
By ribonucleotide reductase Intermediate electron carriers NAPH is reducing agent
How is colour vision mediated
By three cone receptor types who are each most susceptible for a certain wavelength Blue Green Red They also use 11-cis-retinal as chromophore
PhastGel IEF -4-6.5
C-M-R High hazard level (corrosive)
Chronic toxicity
C-M-R Carcinogenic Mutagenic Reproductive toxicity -> not for pregnant women
Trypsin cleavage site
C-terminal of (+) charged side chains (K, R)
Cyanogen bromide cleavage site
C-terminal of INTERNAL M
Chymotrypsin cleavage site
C-terminal of aromatic amino acids (F, W, Y)
C3 plants requre 18 molecules of ATP to synthesize 1 molecule of glucose. C4 plants require 30 molecules of ATP to synthesize 1 molecule of glucose. Why would any plant use C4 metabolism instead of C3 metabolism give that C3 metabolism is so much more efficient?
C4 metabolism allows rubisco to function efficiently even where the temperatures are high, which favor oxygenase activity. Moreover, C4 metabolism allows desert plants accumulate CO2 at night when the temperatures are cooler and water evaporation is not a problem.
ATP synthase name
CF1CF0 ATP synthase
Sphingomyelin
CHNH2 : Esterified with FA CHOH : Phosphoric acid ester -> CH2-CH2-N+-(CH3)3
Dark reaction products
CO2 fixation with help of ATP and NADPH from light reaction Glucose ADP NADP+
Succinyl CoA
Can be obtained from propionyl-CoA which is produced in ß-oxidation of odd numbered fatty acids
Reactions in mitochondria:
Carbamoyl Phosphate Synthetase I: CO2 +NH4+ +2 ATP Carbamoyl Phosphate Ornithine transcarbamoylase: Carbamoyl Phosphate + ornithinecitrulline
Catalytic enzymes of the urea cycle
Carbamoylphosphate synthetase I (CPS1) Ornithine transcarbamylase (OTC) Argininosuccinic acid synthetase (ASS1) Argininosuccinic acid lyase (ASL) Arginase (ARG1)
Glycoproteins
Carbohydrates linked covalently to a peptide chain e.g. Antibodies
Biotin is needed for
Carboxylation steps - donates carbon
Role of pyridoxal phosphate in amino acid reactions
Carrier of amino group Binds to enzyme [pyridoxal-phosphate-dependent aminotransferase] (Schiff's base)
Debranching enzyme
Catalyzes hydrolysis of α-1,6 linkages
Why is chlorophyll an effective light absorbing pigment?
Chlorophyll contains networks of alternating single and double bonds. Such networks allow electrons to resonate and thus not held tightly by a particular atom. This situation allows excitation of the electrons by light energy.
Chlorophyll is a hydrophobic molecule. Why is this property crucial for the function of chlorophyll?
Chlorophyll is readily inserted into the hydrophobic interior of the thylakoid membranes.
Lipid cyclic forms
Cholesterol Steroid hormones Bile acids
Vitamin D
Cholesterol -> 7-dehydrocholesterol
When we move fatty acids around the body, we need various carrier molecules. One of them is albumin for free fatty acids. But when we are moving cholesterol and other things around we are using lipoprotein particles: gave us 5 classes (know what the 5 classes are and what they do)-
Chylomicrons: transport dietary lipids, especially triacylglycerols throughout the bloodstream (dietary-fat transport) ~Very Low-Density Lipoproteins (VLDL): bring triacylglycerol molecules from the liver to cells where the fatty acids are removed by lipases for import into cells, leaving an Intermediate-Density Lipoprotein (IDL). (endogenous-fat transport) ~Intermediate-Density Lipoprotein (IDL) is taken up by the liver and converted into a Low-Density Lipoprotein (LDL) by the removal of more triacylglycerols. (LDL precursor) ~Low-Density Lipoprotein (LDL): major cholesterol carriers in the blood and has apoprotein B-100 on its surface to direct it to specific cells. (cholesterol transport; bad cholesterol) ~High-Density Lipoprotein (HDL): cholesterol scavengers (reverse cholesterol transport= removes cholesterol from macrophages and returns it to the liver for use as bile salts or excretion; good cholesterol)
Proteases examples
Chymotrypsin - cleaves at C-terminal of aromatic amino acids Trypsin - cleaves chymotrypsinogen - cleaves at C-terminal of (+) charged amino acids Caspase 3 - I-CAD Thrombin - cleaves fibrinogen to fibrin
Citrate in anabolism of CAC
Citrate -> Oxaloacetate + Acetyl-CoA Acetyl-CoA into FA synthesis
[Anabolism] CAC lipid anabolism
Citrate -[ATP-citrate lyase]-> OAA + A-CoA Acetyl-CoA into FA synthesis
Control points in CAC
Citrate synthase - inhibited by ATP, NADH, succinyl CoA and citrate Isocitrate dehydrogenase - inhibited by ATP and NADH α-ketoglutarate dehydrogenase - inhibited ba ATP, NADH and succinyl-CoA
ß-oxidation
Cleaves 2 carbon atoms per cycle from carboxyl end of fatty acid 4 reactions with 4 enzymes 1: [Acyl-CoA dehydrogenase] Oxidation of α, β C-C single bond to C=C 2: [Enoyl-CoA hydratase] Hydroxylates β-carbon 3: [1-Hydroxyacyl-CoA dehydrogenase] C=O 4: [Thiolase] Cleaves α-β C-C bond; β carbon (C=O) becomes new acceptor for -S-CoA
Ribonuclease P
Cleaves precursor of tRNA to give functional tRNA
Coenzyme
Cofactor that is an organic molecule Not every co-factor is a coenzyme but every coenzyme is a co-factor
Role of vitamin C for collagen
Collagen is held together by H-BONDS between HYL and HYP Prolyl and Lysyl hydroxylase require vitamin C as cofactor
Super-secondary structures
Combinations of secondary structures give rise to Super-secondary structures (DOMAINS) give rise to Repetitive super secondary structures (MOTIF) - greek key - ß-meander
Reversible inhibitors
Competitive inhibitors Non-competitive inhibitors Uncompetitive inhibitors
Chaotropic compounds
Compounds disrupting H-bonds
Turnover rate
Concentration of substrate disappearing per second [mol/L*s]
Know about Acyl-Malonyl ACP condensing enzyme
Condenses Acetyl ACP and Malonyl ACP
Positive cooperativity hemoglobin
Conformational change induced by O2 binding (β-chains get in closer contact) Less sterical hindrance at Heme-group
Sphingolipids
Contain sphingosine, a long-chain amino alcohol
Bradford assay
Coomassie Brilliant Blue binds to basic sites of protein -> if bound absorption maximum at 595 nm + compatible to reducing agents + high sensitivity - not tolerant to SDS and detergents - small linear range - depends on AA composition (basic AAs)
Biuret assay
Copper in alkali solution forms chelate with peptide chains and forms purple color - low sensitivity - depends on AA composition (aromatic AAs! [TYR])
Folin-Ciocalteu reagent phenol reagent
Corrosive
Sodium hydroxide
Corrosive
Triton X-100
Corrosive Low hazard level
HCl
Corrosive Low hazard level
Know how is acetyl CoA carboxylase regulated
Covalently- by phosphorylation (off), and dephosphorylation (on) Allosterically - by citrate (causes it to polymerize)
Evolutionary implications of photosynthesis
Cyanobacteria were the first bacteria to use H2O as electron donor Earlier photosynthesis reactions might have had other electron donors (H2S) CO2 is acceptor
Know what organisms can fix nitrogen biotically
Cyanobacteria, bacteria (Azotobacter), symbiotic bacteria (Rhizobium
Leukotrienes
Cysteine + triene (3 conjugated double bonds)
Fatty acid biosynthesis place
Cytosol
Lactose
D-galactose D-glucose ß-1,4 Galactose is a C4 epimer of glucose
Sucrose
D-glucose and D-fructose α-1,2
What are the four bases in DNA and RNA?
DNA - A-T C-G RNA - A-U C-G
1) What are DNA polymerase III and DNA polymerase I and what are their enzymatic activities? Where are they used? What is the main one used in replication? Which one is used to get rid of Okazaki fragments? Which activities go with each one?
DNA pol I 5' → 3' polymerization activity (Removes Okazaki fragments) 5' → 3' exonuclease activity (nick translation) 3' → 5' exonuclease activity (proofreading) DNA pol III 5' → 3' polymerization activity (Main polymerase involved in replication) 3' → 5' exonuclease activity (proofreading)
purine biosynthesis
De novo synthesis of IMP requires 7 ATP (!) per molecule IMP (inosine monophosphate) is precursor for AMP and GMP and takes even further ATP Therefore many salvage pathways exist Summary: ribose-5-phosphate from phosphate pentose pathway to IMP (7 ATP) IMP to AMP or GMP
Know about Lesch-Nyhan syndrome.
Defect in hypoxanthine-guanine phosphoribosyl transferase.... Leads to mental defects and self-mutilation behavior
General disadvantage of colorimetric protein quantification
Dependent on AA sequence Biuret, Lowry and BCA: trp,his,tyr,... Bradford: basic side chains
Know how protein phosphatase 1 reverses that and turns on the other pathway while shutting off the first pathway.
Dephosphorylates glycogen phosphorylase, phosphorylase kinase, and glycogen synthase. - Can simultaneously shutdown and start up competing pathways - Inactivated by Protein Kinase A
Cell disruption methods with denaturation of proteins
Detergents Chaotropic compounds Only for cells outside tissues
Enzyme activity assays types
Discontinuous vs continuous Direct vs indirect Product assayed directly or product is further converted into a detectable compound Colorimetric vs fluormetric vs luminometric
Covalent forces stabilizing 3° structure
Disulfid bonds -S-S- between Cysteine residues
Glycogen breakdown
Does NOT need ATP > phosphorolysis, not hydrolysis 1) Glycogen + Pi -[glycogen phosphorylase]-> Remainder + G1P 2) G1P -[phosphoglucomutase]-> G6P
Lineweaver-Burk plot
Double reciprocal graph of Michaelis-Menten equation. Used to determine Vmax of enzymatic substrate conversion
Secondary active transport
Driven by another proton gradient
Aldose
E.g. glucose
Redox-reaction
Electron-transfer Oxidizing agent takes electrons/H-atoms Reducing agent gives up electrons/H-atoms
Know what elongases and Desaturases are
Elongases - enzymes associated with the ER, add 2C units from malonyl CoA to palmitate to produce FAs longer than 16C. Desaturases - Work at the ER, introduce double bonds into FAs (if we don't have the correct desaturates we must consume them [essential])
Phosphofructokinase
Enzyme that catalyzes phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate Key regulatory enzyme of glycolysis: allosterically inhibited by ATP and fructose-1,6-bisphosphate exists in many isozymes with different numbers of M and L subunits
Holoenzyme
Enzyme with prothetic group (active) Apoenzyme: without prosthetic group (inactive)
Enzymes affect kinetics or thermodynamics?
Enzymes do not affect thermodynamics - do not shift the equilibrium Enzymes affect kinetics - lower reaction energy and accelerate attainment of equilibrium -> same equilibrium but reached much faster
Know how hormones like epinephrine and glucagon effect glycogen utilization, know about regulation of the enzymes in glycogen utilization
Epinephrine - promotes phosphorylation of b to a mostly mobilizes glucose in the muscles. Glucagon works to mobilize glucose in the liver.
ΔG=0
Equilibrium
NAD+/NADH structure
Ester bond between 2 phosphate groups each phosphate is bound to a ribose molecule (5') one ribose is bound to and adenine (1') one ribose is bound to nicotinamide ring (1')
Site of ATP formation
F1 unit of ATP synthase
Energy yield carbohydrates vs fatty acid
FA: 9 kcal/g C: 4 kcal/g
First step of FA oxidation
Fatty acid activation step 1: ATP is consumed, AMP bound to fatty acid step 2: CoA-SH replaces AMP RCO-S-CoA
Lipid open chain forms (9)
Fatty acids Triacylglycerols Sphingolipids Phosphoacylglycerols Glycolipids Lipid soluble vitamins Protaglandins Leukotrienes Thromboxanes
Oxygen binding of Heme
Fe2+-O=O 120°
Ketones general structure characteristic functional group
Feature a carbonyl group -> R' instead of an H (aldehydes)
Aldehydes general structure characteristic functional group
Feature a carbonyl group -> with an H
Thiols general structure characteristic functional group
Feature a sulfhydryl group (SH) e.g. CH3-SH -> cysteine
Carboxylic acids general structure characteristic functional group
Features a carboxyl group -> like a carbonyl group but hydroxyl group (OH) instead of H or R'
Amides general structure characteristic functional group
Features an amide group Like ketone but R' is an NH2, NHR or NR2
Esters general structure characteristic functional group
Features an ester group O II -C-O-R
Phosphoric acid anhydrides general structure characteristic functional group
Features phosphoric anhydride group O O II II R-P-O-P-R' I I OH OH
Phosphoric acid esters general structure characteristic functional group
Features phosphoric ester group
Know about regulation of purine and pyrimidine biosynthesis, overall/how it's done
Feedback inhibition
Pyrimidine biosynthesis regulation
Feedback inhibition
Roti-Quant
Flammable Corrosive
1-butanol
Flammable Corrosive Low hazard level
Bacillol
Flammable Corrosive Low hazard level
Ethanol
Flammable Low hazard level
Acetic acid
Flammable Corrosive to metals Low hazard level
Acetone
Flammable Low hazard level
Ketone bodies
Form if Acetyl-CoA cannot be used -> starvation, more fats than glucose intake, diabetes Acetone Acetoacetate D-3-hydroxy-n-butyric acid
Feedback inhibition
Formation of product inhibits its continued production
Positive reaction (change in energy)
Free overall energy increases ("up-hill") -> reaction will not occur without energy supplied from external source
Glycogen synthesis
G1P + UTP -> UDPG + pyrophosphate UDPG can be added to the growing glycogen chain by glycogen synthase Catalyzes α-1,4 linkages Branching enzyme takes 7 unit polymer from growing branch and forms a new α-1,6 linked branch
Pentose-Phosphate pathway oxidation steps
G6P [NADP+ -> NADPH] 6-Phosphogluconate [NADP+ -> NAPH] Ribulose-5-phosphate
Why is it physiologically advantageous for the pancreas to use GLUT2 with a high Km value as the transporter that allows glucose entry into Beta cells?
GLUT2 transports glucose only when the blood concentration of glucose is high which is precisely the condition in which the beta cells of the pancrease secrete insulin.
GenTG/ GenTSV
Genetically modified Os Risk assessment
Glutamic acid 3- and 1-letter code
Glu E
Major hormones regulating carbohydrate metabolism
Glucagon Insulin Epinephrine
Regulation based on kinases and phosphatases - make sure you know it! How hormones will affect those.
Glucagon - glucose is gone need to mobilize glycogen stores. Epinephrine - need energy Insulin - time to store carbon
Why is it advantageous for the liver to have both hexokinase and glucokinase to phosphorylate glucose?
Glucokinase enables the liver to remove glucose from the blood when hexokinase is saturated ensuring that glucose is captured for later use.
In the liver and the pancreas, hexokinase and glucokinase phosphorylate glucose. Glucokinase is active only when the blood levels of glucose are high. How might glucokinase differ kinetically from hexokinase so as to function only at high glucose levels?
Glucokinase has a higher Km value which allows this enzyme to become more active at high glucose concentrations conditions that saturate hexokinase.
Although both hexokinase and phopsphofructokinase catalyze irreversible steps ion glycolysis and the hexokinase catalyzed step is first, phophofructokinase is nonetheless the pacemaker of glycolysis. What does this information tell you about the fate of the glucose 6-phosphate formed by hexokinase?
Glucose 6-phosphate must have other fates. Indeed it can be converted into glycogen or be processed to yield reducing power for biosynthesis.
Glycogen
Glucose subunits α-1,4 and α-1,6
Which glycolysis steps are irreversible and are not reversed in gluconeogenesis?
Glucose to G6P F6P to F-1,6-BP PEP to Pyruvate
Starting step urea cycle
Glutamate (ammonia carrier) + acetyl-CoA -> N-acetyl-glutamate N-acetyl-glutamate allosterically activates CPS1 for the formation of carbayl phosphate
What is important about glutamate dehydrogenase and glutamine synthase what are they important for
Glutamate dehydrogenase combines alpha ketogluterate and ammonia to form glutamate using the reducing power of NADPH or NADH. Glutamine synthetase then adds another ammonia to glutamate to form glutamine using ATP hydrolysis to drive the reaction. This is important because it is used as a nitrogen doener in many biochemical reactions.
Amino acid families (6)
Glutamate family Aspartate family Serine family Pyruvate family Aromatic family Histidine family
Phosphatidyl ester
Glycerol with three residues (esterified) - R1 (fatty acid) - R2 (fatty acid) - phosphoric acid ester with R3 (POR3) R3 can have multiple forms e.g. phosphatidylcholine
Shuttle mechanisms
Glycerol-Phosphate Shuttle (1,5 ATP/NADH) Less efficient Malate Aspartate Shuttle (2,5 ATP/NADH) More efficient Carry NADH produced in glycolysis into mitochondrium for oxidative phosphorylation
Non-polar amino acids (9)
Glycine Alanine Phenylalanine Valine Leucine Isoleucine Proline Tryptophan Methionine
3 enzymes to break it down (gluycogen): glycogen phosphoralase, glycogen debranching enzyme, phosphogluco mutase - know what they do.
Glycogen Phosphorylase cleaves the α 1 4 main chain glycosidic bond by adding inorganic phosphate (Pi) to form glucose 1-phosphate and a glycogen chain shorter by one glucose. Glycogen Debranching Enzyme is a bifunctional enzyme and works on the 4 residue "nub" left at branchpoints by Glycogen Phosphorylase. The transferase activity removes 3 residues from the "nub" and moves it to a non-reducing end of a main chain forming an α 1 4 glycosidic bond. Phosphoglucomutase converts glucose 1-phosphate into glucose 6-phosphate
Why store glucose as glycogen? How does that work in with number of non-reducing ends? Difference between glycogen and starch, which has more, which is more branched. What does having more non-reducing ends do for you? Why is this good?
Glycogen is readily mobilized, much more so than fatty acids. Therefore, glycogen is a quick source of energy instead of a long-term resource. -Glycogen stores also provide the brain with glucose in between meals. -Glycogen has more branches than starch -Having more non-reducing ends makes synthesis and degradation a rapid process.
Glycogen degrading enzyme
Glycogen phosphorylase
Some of the early research on glycolysis was supported by the brewing industry. Why would the brewing industry be interested in glycolysis?
Glycolysis is a component of alcoholic fermentation the pathway that produces alcohol for beer and wine. The belief was that understanding the biochemical basis of alcohol production might lead to a more efficient means of producing beer,.
Glycolysis energetics
Glycolysis is exergonic
purine salvage - guanine
Guanine + PRPP -[hypoxanthine guanine phosphoribosyltransferase]-> GMP
Molecules affecting O2 binding capacity of Hb
H+ CO2 BPG
Buffer in blood
H2CO3/HCO3-
Reaction PSII
H2O is oxidized to 1/2 O2
Buffer in cells
H2PO4-/HPO42-
ELISA Signal detection by enzyme-conjugated ABs
HRP - horseradish peroxidase Substrate pNPP (p-nitrophenylphosphat) aP - alkaline phosphatase Substrate OPD (o-phenylenediamine)
CO2 fixation in tropical plants
Hatch-Slack Pathway/C4-pathway
If glucose-6-phosphate dehydrogenase is not active enough, you won't have enough NADP. This will lead to some diseases:
Hemolytic anemia- unable to make sufficient NADPH (RBCs are susceptible to oxidative damage) Heinz bodies - form in the absence of glutathione and cause RBCs to lyse. Can also be a plus, if you have a parasite that needs the host cells (glucose 6 phosphate dehydrogenase enzyme) to be working and it's not working, can provide protection against the parasite. Protection against malaria
Glycosaminoglycans
Heparin Hyaluronic acid Chondroitin sulfate & keratan sulfate Consists of disaccharide subunits of 1) amino sugar 2) negatively charged due to carboxylic acid group
Lipid definition
Heterogenous class of naturally occurring organic compounds -> insoluble in water -> amphipathic Open chain forms and cyclic forms
Enzymes Glycolysis
Hexokinase (G6P) Phosphoglucose isomerase (F6P) Phosphofructokinase (F-1,6-BP) Aldolase (dihydroxyactone + glyceraldehyde-3P) Triose phosphate isomerase (Glyceraldehyde-3P) Glyceraldehyde-3-phosphate dehydrogenase (1,3-BPG) Phosphoglycerate kinase (3-PG) Phosphoglycerate mutase (2-PG) Enolase (PEP) Pyruvate kinase (pyruvate)
Physical methods of microbial control
High T (dry vs moist heat) Low T Filtration Radiation Osmotic pressure
Why do high concentrations of CO2 inhibit photorespiration?
High concentrations of CO2 prevent O2 from entering the active site of rubisco.
Dichloromethane
High hazard level (chronic) -> C-M-R
cAMP mediated signal transduction
Hormone binds to hormone receptor G protein dissociates: GTP is bound to α-subunit and travels to adenylate cyclase, ß:γ subunits adenylate cyclase is activated and catalyzes ATP -> cAMP + PP until GTPase activity of α-subunit hydrolyzes GTP Then G protein reassociates and adenylatecyclase is inactive again cAMP catalyzes the activation of a protein kinase (eventually kinase cascade and crosstalk) Target protein is phosphorylated (could be a TF, enzyme, structural proteins etc)
Epstein-Barr Virus
Human Herpes Virus 4 Burkitt's lymphoma nasopharyngeal carcinoma mononucleosis Enveloped (inactivated by heat) 90% of population infected
Non-covalent forces stabilizing 3° structure
Hydrogen bonds between polar AA residues Hydrophobic interactions between non polar AA residues Hydrophobic interactions between AA residues of opposite charge Electrostatic repulsion between AA residues of same charge
How is endergonic energy provided to enable (non-spontaneous ΔG>0) biochemical reactions?
Hydrolysis of ATP ATP -(-H2O)-> ADP+Pi Releases 7.3 kcal/mol ATP
Main force driving formation of lipid bilayers
Hydrophobic interactions
What are the reactibve oxgen species and why are they especially dangerous to cells?
Hydroxyl radical hydrogen peroxide superoxide ion and peroxide. These small molecules react with a host of macromolecules including proteins nucleotides and membranes to disrupt cell structure and function.
purine salvage - hypoxanthine
Hypoxanthine + PRPP -[hypoxanthine guanine phosphoribosyltransferase]-> IMP
Know about the bifurcating pathway leading to AMP/GMP, common pathway. Not each step, just main difference in regards to PRPP
IMP inhibits glutamine phosphoribosyl amidotransferase, which both AMP and GMP inhibit
How does the inhibition of ATP-ADP translocase affect the citric acid cycle? Aerobic glycolysis?
If ADP cannot enter the mitochondria, the electron transport chain will cease to function because there will be no acceptor for the energy., NADH will build up in the matrix. Recall that NADH inhibits some citric acid cycle enzymes and NAD+ is required by several citric acid cycle enzymes. Glycolysis will stop function aerobically but will switch to anaerobic glycolysis so that the NADH can be re-oxidized to NAD+ by lactate dehydrogenase.
If actively respiring mitochondria are exposed to an inhibitor of ATP-ADP translocase, the electron transport chain ceases to operate. Why?
If ATP and ADP cannot exchange between the matrix and the mitochondria ATP synthase will cease to function because its substrate ADP is absent. The proton gradient will eventually become so large that the energy released by the electron transport chain will not be great enough to pump protons against the larger than normal gradient.
Peroxide of vegetable oil
If oil gets old it is peroxidized We check for peroxidation
Years ago it was suggested that uncoplers would make wonderful diet drugs. Explain why this idea was proposed and why it was rejected. Why might the produces of antiperirants be supportive of the idea?
If oxidative phosphorylation were uncoupled no ATP could be produced. In a futile attempt to generate ATP much fuel would be consumed the danger lies in the dose. Too much uncoupling would lead to tissue damage in highly aerobic organs such as the brain and heart which would have sever consequences for the organism as a whole. The energy that is normally transformed into ATP would be released as heat. To maintain body temperature sweating might increase although the very process of sweating itself depends on ATP.
If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron transport chain ceases to operate. Why?
If the proton gradient cannot be dissipated by flow through ATP synthase the proton gradient will eventually become so large that the energy released by the electron transport chain will not be great enough to pump protons against the larger than normal gradient.
What is the mechanistic basis for the observation that the inhibitors of ATP synthase also lead to an inhibition of the electron transport chain?
If the proton gradient is not dissipated by the influx of protons into a mitochondria with the generation of ATP the outside of the mitochondria eventually develops such a large positive charge that the electron transport chain can no longer pump protons against the gradient.
Carnitine
Important for transport across mitochondrial membrane 1) Acetyl-CoA crosses outer membrane 2) Carnitine acyltransferase in intermembranen space transfers acyl to carnitine 3) Acyl-carnitine crosses inner membrane 4) Carnitine acyl transferase retransfers fatty acid to CoA-SH RCO-S-CoA goes into ß-oxidation
Vitamin K
Important in blood clotting Possesses REPEATING isoprene units which are unsaturated hydrocarbon units
Who is Rosalind Franklin and why are scientists ticked off that she got screwed out of a Nobel Prize?
In 1953, Watson and Crick published there ground-breaking paper describing the structure of DNA (Figure 32.1), for which they received the Nobel Prize. They used X-Ray crystallography data of Rosalind Franklin in their analysis, and they received this data by questionable means. Franklin did not share in the Nobel Prize, and this is one of the major embarrassments in Science. Essentially, Franklin's data was stolen and she got hosed.
Glyoxylate pathway
In plants and some bacteria Modification of CAC Skips the 2 oxidative decarboxylations in and routes via glyoxylate Helps plants to grow in the dark: Plants rich in lipids ß-oxidation + glyoxylate pathway
*Know about RNA processing, . This is different in eukaryotes why?-where does transcription and translation occur in prokaryotes and where do those processes occur in space and time in eukaryotes:
In prokaryotes, mRNA molecules can be translated as soon as they are produced and require no further processing. In fact, translation may start before transcription is even finished. For rRNA and tRNA molecules, however, post-transcriptional modifications are usually required. Since prokaryotes do NOT have a nucleus, replication, transcription, and translation all take place in the cytoplasm. In Eukaryotes replication and transcription occur in the nucleus, and the mature mRNA molecules are translated in the cytoplasm. Therefore, there is a spatial and temporal disconnect between transcription and translation in eukaryotes that is not seen in prokaryotes. This disconnect between transcription and translation also enables eukaryotes to regulate transcription and translation much more finely than prokaryotes are able to.
What is the difference in fatty acid synthase in mammals and bacteria? Mammals have a large peptide that dimerizes, bacteria have individual genes that make individual peptides that come together. Why is each one benefical or detrimental?
In the bacteria if the genes are interupted then the membrane won't properly form so the organism will die. In animals there are check points to avoid complications. This can lead to tumor formation though.
Distinguish between a closed promoter complex and an open promoter complex
In the closed promoter complex, the DNA is in the form of a double helix. In the open promoter complex, RNA polymerase has separated the two DNA strands in order to expose the template, and RNA synthesis has begun
Zymogen
Inactive enzyme precursor
Why are bands broader for smaller molecules in SEC
Increased diffusion because of longer time spent in column Pass detector more slowly -> need to be normalized
Oxytocin
Induces labor in pregnant women and controls contraction of the uterus
How do these hormones (insulin) effect the regulation of the kinases and phosphatases in glycogen mobilization or glycogen storage?
Insulin - Causes a phosphorylation cascade that results in phosphorylation of Glycogen Synthase Kinase, inactivating it (making glycogen synthase NOT being phosphorylated, so glycogen is synthesized.) - Also increases the number of glucose transporters in the plasma membrane
IRS
Insulin receptor substrate - is phophorylate by phosphorylated Tyr residues of β subunits of insulin receptor
Control of Adenylate Cyclase
Interplay of stimulatory and inhibitory G proteins Stimulatory G protein: Gs - receptor: ß Inhibitory G protein receptor: Gi - receptor α2
Amphibolic
Involved in both ana- and catabolic reactions (CAC is amphibolic)
Oxygen evolving process
Involves PSII through five oxidation states 2 H2O -> O2 + 4 H+
Strongest non covalent interactions
Ionic interactions (depends) 1-20 kcal/mol Hydrogen bonds 5 kcal/mol
Transferrin
Iron (Fe3+) transport protein in blood Carries sialic ACID containing compounds The more sialic acid is supposed to be loaded (on the different types) the lower pI Transferrin is tested to analyze genetic diseases and to detect regular alcohol abuse
Insulin activation
Is a peptide hormone and has an inactive precursor (PROINSULIN) Removal of residues 31-65 yield A and B chain
Which enzymes are the key regulatory enzymes of the citric acid cycle itself?
Isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase.
Amino acids that contain a second stereocenter
Isoleucine and threonine
What is the cheif benefit of being able to perform the glyoxylate cycle?
It enables organisms such as plants and bacteria to convert fats through acetyl CoA into glucose.
Why is dinitrogen so stable, how do we reduce it to NH3 or NH4+
It is so stable because it has a triple bond. We can reduce it via the haber baush process or lightning strikes or via free living bacteria.
Ribonucleotide reductase. Know what it does, how it does it, how it is regulated and why it is a gutsy move on the cells part.
It works on all four ribonucleotide disphosphates using the reducing power of NADPH. It uses a free radical so it is tightly controlled (dangerous)
Malate aspartate shuttle
L-Malate and L-Aspartate can cross the mitochondrial membrane while NADH cannot In cytosol L-Malate -[transamination]-> Oxaloacetate Oxaloacetate is then reduced to L-Aspartate (NADH -> NAD+) L-aspartate is transported inside In matrix L-Aspartate is transaminated to oxaloacetate Oxaloacetate is then oxidized (NAD+ -> NADH) to L-Malate again L-Malate is transported outside 2,5 ATP/NADH
Why is the conversion of lactic acid from the blood into glucose in the liver in an organisms best interest?
Lactic acid is capable of being further oxidized and is thus useful energy. The conversion of this acid into glucose saves the carbon atoms for future combustion.
Leucine 3- and 1-letter code
Leu L
Alkenes general structure characteristic functional group
Like an alkane but double bond: C=C double bond
Alcohols general structure characteristic functional group
Like an alkane but feature an hydroxyl (OH-) group
ELISA Competitive immuno assay
Limited amount of ABs -> tested antigen is labelled Tested antigen competes with labeled antigen for immobilized antibodies The more tested antigen the less signal + low amount of tested antigen results in a strong signal, still
Know what lipase and Perilipin A do.
Lipases - Remove the FAs from the triacylglycerols, yielding 3 free FAs and a molecule of glycerol Perilipin A - When phosphorylated, it restructures fat droplets making triacylglycerols more accessible to lipase.
Muscle and liver - relative amounts of glycogen on a per week basis
Liver- higher concentration of glycogen than muscle, but more total glycogen in muscle than in liver.
Relationship between protein MW and relative electrophoretic mobility in SDS-PAGE
Logarithmic
Consequences of wrong protein folding
Loss of function Aggregates of protein
8-Hydroxychinoline
Low hazard level
Trichloroacetic acid
Low hazard level Corrosive Harmful to environment
Bohr effect
Lower pH decreases Hb's affinity for oxygen (because H+ binds to several residues of globin chains) -> in metabolically active tissues (due to high CO2 concentration) the pH is lower (7.2) -> here oxygen release is facilitated
Lysine 3- and 1-letter code
Lys K
Basic amino acids (3)
Lysine Arginine Histidine
Know the structure of glycogen- main chain and branch point glycosidic linkage
Main chain - α 1-4 glycosidic bonds Branchpoint linkages - α 1-6 (occur every 10 main chains)
Biotin assisted reactions we know
Malonyl-CoA -> Acetyl-CoA (FA synthesis) Pyruvate -> Oxaloacetate (Gluconeogenesis)
Chemiosmotic coupling - how is it mediated
Membrane protein of the inner mitochondrial membrane are not symmetrically oriented within the membrane Since the electron transport is a chain of redox reactions with decreasing energy H+ are accepted and donated When membrane complex is reduced it takes up H+ from matrix When membrane complex then is oxidized again it releases the proton into the intermembrane space
Make sure you can recognize (multiple choice) modes 1,2,3,4 as what is needed most by the cell.
Mode 1 - More Ribose 5-phosphate is needed than NADPH Mode 2 - Ribose 5-phosphate and NAPH needs are balanced (oxidative part of PPP is used) Mode 3 - More NADPH is needed than Ribose 5-phosphate Mode 4 - Both ATP and NADPH are needed (oxidative part of PPP is used to produce NADPH, and glycolytic pathway is used to produce ATP from ribose 5-phosphate).
Catalytic antibodies
Molecular engineered antibodies which stabilize a transition state of a reaction -> CATALYSIS
Receptor Tyrosine kinases Class I
Monomeric 2 cysteine-rich domains on outside part tyrosine kinase portion inside
The degradation of the aromatic amino acids requires the use of oxygenases to break the aromatic ring
Monooxygenase - Only one O2 ends up in the product (other in H2O) Dioxygenase - Uses both O2
Liver is primarily a gluconeogenic tissue, wheras muscle is primarily glycolytiv. Why does this division of labor make good physiological sense?
Muscle is likely to produce lactic acid during contraction. Lactic acid is a strong acid and cannot accumulate in muscle or blood. Liver removes the lactic acid from the blood and converts it into glucose. The glucose can be released into the blood or stored as glycogen for later use.
Chitin
N-actyl-ß-D-glucosamine subunits ß-1,4 linked Makes up exoskeleton of invertebrates and cell walls of some fungi, algae and yeasts
XC - Know the stoichiometry of Nitrogen fixation
N2 + 8e- + 8H+ + 16ATP + 16H2O -> 2 NH3 + H2 + 16 ADP + 16Pi
Glycerol Phosphate Shuttle
NADH cannot cross the mitochondrial membrane while glycerol phosphate and dihydroxyacetone phosphate can Dihydroxyacetone phosphate is reduced to Glycerol phosphate (NADH -> NAD+) Glycerol phosphate is transported into the mitochondrion Glycerol phosphate is oxidized to dihydroxyacetone phosphate (FAD+ -> FADH2 again Dihydroxyacetone is transported outside 1,5 ATP/NADH
Reaction PSI
NADP+ is reduced to NADPH
Identify the ultimate electron acceptor and the ultimate electron donor in photosynthesis. What powers the electron flow between the donor and the acceptor?
NADP+ is the acceptor. H2O is the donor. Light energy powers the electron flow.
BioStoffV
Natural biological working materials WITHOUT infectious sensitizing nor toxic effects
What is important about nitrogenase reductase and nitrogenase what does each one do
Nitrogenase reductase brings in high energy electrons to the nitrogenase enzyme so that it can reduce the dinitorgen triple bond. Nitrogenase actually reduces N2 to NH3.
Size Exclusion chromatography vs SDS-PAGE
No denaturation; bigger molecules elute FIRST
Ribozymes
Non protein biocatalysts RNA
Why are the reactions of the Calvin cycle soometimes referred to as the dark reactions? Do they take place only at night or are the a grim secret reaction?
Nothing grim or secret about these reactions, they are called the dark reactions because they do not directly depend on light.
Know the difference between a nucleoside/tide
Nucleoside - Nitrogenous base on ribose or deoxyribose Nucleotide - Nitrogenous base on ribose or deoxyribose AND 1+ phosphate group *T is after S, carries 1 more thing
Inosine
Nucleoside with hypoxanthine
Turnover number
Number of moles a substrate that reacts to form product per mole of enzyme per unit of time Also called catalytic constant Turnover number corresponds to physiological demands on the enzyme
ß anomer
OH on anomeric carbon is CIS to terminal CH2OH
α anomer
OH on anomeric carbon is TRANS to terminal CH2OH
Ceramide
ONE fatty acid linked to sphingosine by an amide bond
What happens to 3-phosphoglycerate after fixation
Of 12 formed 3PG molecules, 10 are used to recover ribulose-1,5-bisphosphate 2 are used to form one molecule of glucose
What is the fate of oxaloacetate after split from acetyl CoA by ATP-citrate lyase
Oxaloacetate -[Malate dehydrogenase]-> Malate -[Malic enzyme]-> Pyruvate -> replaces NADH with NAPH
If plants are illuminated by a combination of light of 680 nm and 700 nm the oxygen production exceeds that of either wavelength alone. Explain.
Oxygen consumption will be maximal when photsystems 1 and 2 are operating cooperatively. Oxygen will be efficiently generated when electrons from photosystem 2 fill the electron holes in photosystem 1 which were generated when the reaction center of phostosystem 1 was illuminated by light of 700 nm.
Insulin function
PRIMARY: x Glucose absorption into cells SECONDARY: x Glycogen breakdown x Glycolysis x FA synthesis
Cyclic electron transport
PSII not involved: No water is split and no NADPH is produced Electrons are transported back and forth between PSI and Cyt b6-f complex Drives inward transport of H+ by Cyt b6-f complex Plastocyanin is docked to PSI ATP synthase uses energy of proton gradient for phosphorylation of ADP
Antibody structure
Paratope = Variable portion Fc portion = Constant portion 2 identical heavy chains 2 identical light chains Bound by disulfide bridges ABs are glycoproteins
Insulin structure
Peptide hormone A and B chain linked by disulfide bridges
Bacterial cell walls
Peptidoglycan polysaccharide NAM-NAG ß-1,4 N-acetyl-muramic acid B-acetyl-glucosamine
Types of membrane protein anchoring
Peripheral proteins Bound by electrostatic interactions Integral proteins Bound tightly to membrane interior N-myristoyl anchoring motif anchored by N-terminal glycine S-palmitoyl anchoring motif anchored by cysteine residue
Phenylalanine 3- and 1-letter code
Phe F
*Know about diseases or maladies associated with deficiencies in amino acid degradation pathways:
Phenylketonuria- disease of phenylalanine degradation pathway. Others that may need to be able to recognize: citrullinema, tyrosinemia, albinism, homocystinuria, hyperlysinemia
Chlorophyll reaction center structure
Pheophytin Menaquinone (QA) CoQ (ubiquinone) Chlorophyll RESONANCE energy transfer
Experiment reaction with alkaline phosphatase
Phosphatase removes phosphate Artificial substrate Paranitrophenol-phosphate -alkaline-phosphatase-> Paranitrophenol + phosphate Inhibited by phosphate -> feedback inhibition
Nucleotide
Phosphate + Sugar + Base
PIP2
Phosphatidylinositol 4,5-bisphosphate
Biological membrane
Phospholipid bilayer Hydrophobic fatty acid residues stick together inside membrane Polar head groups face inner and outer aqueous environment
PRPP (formation)
Phosphoribosyl-pyrophosphate from ribose 5-phosphate + ATP
What does PKC do in PIP2 mediated signal transduction?
Phosphorylates other target proteins Phosphorylates Ca2+ channels for Ca2+ influc - prolonged Ca2+ effect even if intracellular reservoir in ER are spent
Know how kinases will turn on one set of competing reactions and turn off the other set with glycogen utilization and glycogen synthesis.
Phosphorylation -Glycogen synthase - a to b form (inactivates) -Glycogen phosphorylase - b to a form (activates)
What is the significane of photoinduced separatation of charge in photosynthesis?
Photo induced separation of charge results when a high energy electron generated by light absorption moves to a neighboring acceptor molecule with a lower excited state. This step is in photosynthesis is fundamental because the now negatively charged acceptor molecule possess a high energy electron that can be used to generate a proton gradient of biosynthetic reducing power.
What is photorespiration, what is its cause, and why is it believed to be wasteful?
Photorespiration is the consumption of oxygen by plants with the production of CO2 but it doesn't produce energy. Photorespiration is due to the oxygenase activity of rubisco. It is wasteful because instead of fixing CO2 for conversion into hexoses rubisco generates CO2.
Photosynthesis can be measured by measuring the rate of oxygen production. When plants are exposed to light of wavelength 680 nm more oxygen is evolved than if the plants are exposed to light of 700 nm. Explain.
Photosystem 2 in conjunction with the oxygen generating complex powers oxygen release. The reaction center of photosystem 2 absorbs light maximally at 680 nm.
Phosphoric ester
Pi comes from ATP Phosphoric ester at OH group of 6' carbon example: glucose-6-phosphate
H2O polarity
Polar bonds: O (δ-) has higher electronegativity than Hs (δ+) Therefore side of O is negatively charged and molecule is polar
Mode of TLC
Polar stationary phase Non-polar mobile phase (normal phase) -> polar analytes are retained more strongly
Cellulose
Polysaccharide composed of 2800 D-glucose units ß-1,4 linked Extensive H-bonding Cellubiose disaccharide subunits
Know what prenylation of proteins is
Posttranslational modification
It has been noted that the mitochondria of muscle cells often have more cristae than the mitochondria of liver cells. Provide an explanation for this observation.
Presumable because the muscle has greater energy needs especially during exercise so it will require more ATP. This requirement means that more sites of oxidative phosphorylation are called for and these sites can be provided by an increase in the amount of cristae.
ELISA Signal amplification via biotinylation
Primary AB Secondary AB (biotinylated) Biotinylation binds avidin/streptavidin Complex forms
Proline 3- and 1-letter code
Pro P
Differential Centrifugation
Procedure for separating cellular components according to their size and density by spinning a cell homogenate in a series of centrifuge runs. After each run, the supernatant is removed from the deposited material (pellet) and spun again at progressively higher speeds. Low speed: Nuclei and broken cells are pelleted down Higher speed: Mitochondria Higher speed: Small fragments, ribosomes
Pentose-phosphate pathway serves which function?
Production of 5 carbon sugars for nucleic acid synthesis Production of NADPH
5 C fatty acid (breaks down until 5), break that down, you peel off acetyl CoA, and something else. What is the something else and how it's metabolized.
Propionyl CoA - metabolized by propionyl carboxylase (adds a carbon)
Why protein conformation is important
Proteins are not isolated Bind and interact with other molecules Interactions are guided by non-covalent bonds Interaction can be very specific -> basis of AB specificity -> basis of enzyme specificity -> basis of receptor-ligand binding
Most important co-factor in AA metabolism
Pyridoxalphosphate
Vitamin B
Pyridoxine
Pyrimidine & Purine
Pyrimidines: C, T, U Purines: A, G
Initiation step of citric acid cycle
Pyruvate -> Acetyl-CoA CoA-SH -> CO2 NAD+ -> NADH + H+ Catalyzed by pyruvate dehydrogenase complex (Mg2+ needed as cofactor)
Identify the oxidant and the reductant in the following reaction. Pyruvate + NADH + H+ -><- lactate + NAD+
Pyruvate accepts electrons and is thus the oxidant. NADH gives up electrons and is the reductant.
Gluconeogenesis - First step - ATP??????????????????
Pyruvate is CARBOXYLATED to Oxaloacetate Catalyzed by pyruvate carboxylase Subject to allosteric control: activated by Acetyl-CoA Needed: Mg2+, CO2, ATP, Acetyl-CoA
Gluconeogenesis enzymes for irreversible steps
Pyruvate to PEP via oxaloacetate: Pyruvate carboxylase, PEP carboxykinase F-1,6-BP to F6P: Fructose-1,6-Bisphosphatase G6P to Glucose G6P Phosphatase
Glycogen phosphorolase - B and A form, how to go from B to A, which one favors T/R state?
R - phosphorylase a T - phosphorylase b Glucose binding shifts the equilibrium significantly in the direction of the T state, inactivating the enzyme. ***energy charge controls the switch
Enzymatic hydrolysis of triacylglycerols product
R1COO- R2COO- R3COO- Ionized fatty acid + glycerol
Saponification of triacylglycerols product
R1COO- Na+ R2COO- Na+ R3COO- Na+ Sodium salts of fatty acid + glycerol
P/O
Ratio of molecule of ATP phosphorylated per molecule of oxygen consumed
If you break down a fatty acid via b oxidation, know what the reactants and products are for breaking it down.
Reactants: Fatty acid, lipase?? Products: Acetyl CoA, NADH, FADH2, FA (2 Carbons shorter)
PIP2/Ca2+ mediated signal transduction
Receptor coupled to G protein (α:β:γ) When hormone binds (dissociation of G protein) α:GTP activates Phospholipase C Phospholipase C hydrolyzes PIP2 to IP3 and DAG IP3 triggers the release of Ca2+ from intracellular reservoirs in the ER DAG remains in membrane activating membrane-bound protein kinase C (PKC) PKC phosphorylates Ca2+ channels which then allow Ca2+ influx Ca2+ induces cellular response -> action of DAG -> PKC prolongs cell response
Insulin receptor
Receptor tyrosine kinase class II α2β2 α subunits bind to insulin molecules β subunits span the membrane and AUTOPHOSPHORYLATE tyrosine residues These in turn phosphorylate insulin receptor substrates (IRSs) which act as second messengers in the cell
Red-green-blindness
Red cone and green cone gene are on same chromosome Wrong crossing over can lead to loss of one gene or to gene hybrids
Effect of cholesterol on membranes
Reduce fluidity by stabilizing hydrocarbon tails of fatty acids
BCA assay
Reduction of Cu2+ ions to Cu+ ions in the presence of proteins (bind to peptide bonds) Cu+ ions bind to Bicinchonate (purple colour) + high sensitivity + tolerant to detergents - not tolerant to reducing agents - depends on AA composition (aromatic AAs [TYR]) - expensive
NAD+/NADH red/ox
Reduction: One H+ from benzol ring is given up NAD+ reduced NADH oxidized
Vitamin D role
Regulation of calcium and phosphorus metabolism
Calvin cycle fixation step
Ribulose-1,5-Bisphosphate + CO2 + H2O -> 2 x 3-phosphoglycerate Short-lived intermediate Catalyzed by Ribulose-1,5-Bisphosphate carboxylase oxygenase (RuBisCO)
Fixation step enzyme
Ribulose-1,5-bisphosphate carboxylase oxygenase
Example that contains nitrogenase
Root nodules of leguminous plants
Suggest a reason why rubisco might be the most abundant enzyme in the world.
Rubisco catalyzes a crucial reaction, but it is highly inefficient. Consequently it is required in large amounts to overcome its slow catalysis.
Isoelectric focussing
Separation based upon different pI PAA-gel with covalently bound charged functional groups establishes a pH gradient Proteins migrate to pH=pI -> uncharged
Serine 3- and 1-letter code
Ser S
Hydroxylated AAs
Ser Tyr Thr
Polar amino acids (6)
Serine Threonine Tyrosine Cysteine Asparagine Glutamine
HbA vs HbF
Serine 143 in a instead of Histidine in a γ-chain This results in decreased affinity for BPG
Michaelis-Menten diagram
Shows enzyme activity in relation to substrate concentration KM : substrate concentration of Vmax/2
Chlorophyll structure
Similar to heme group of Mb, Hb and cytochromes BUT Mg2+ ion instead of Fe Hydrophobic side chain anchoring chlorophyll within membrane Tetrapyrrole ring structure
ΔG<0
Spontaneous EXERGONIC energy is RELEASED
The Calvin cycle can be though of as taking place in three stages. Describe the stages.
Stage 1 is the fixation of CO2 with ribulose 1,5 bisphosphate and the subsequent formation of 3 phosphpglycerate. Stage 2 is the conversion of some of the 3-phosphoglycerate into hexose. Stage 3 is the regeneration of ribulose 1,5 bisphosphate.
When are ketone bodies formed?
Starvation: Gluconeogenesis takes away malate from CAC -> acetyl-CoA accumulates and is converted into ketone bodies
Enantiomers
Stereoisomers that are mirror images of each other
5) Know how the STEC (Streptomycin, Tetracycline, Erythromycin & Chlorampenicol) affect protein synthesis at the ribosome (they all affect the ribosome somehow).
Streptomycin (inhibits initiation and cause the misreading of mRNA in bacteria), Tetracycline (binds to the small ribosomal subunit of bacteria and inhibits the binding of aminoacyl-tRNAs), Erythromycin (Binds to the large ribosomal subunit bacteria and inhibits translocation) and Chloramphenicol (inhibits the peptidyl transferase activity of the large ribosomal subunit in bacteria)
Site of dark reactions
Stroma of thylakoid
Pasteurization
Sub-boiling Ts Does NOT STERILIZE liquids but reduces microbial loads
KM
Substrate concentration where Vmax/2 is established Corresponds to binding capacity low KM = high binding capacity high KM = low binding capacity
Isoprene
Subunit for terpenes and cholesterol
How is succinate dehydrogenase unique compared with the other enzymes in the citric acid cycle?
Succcinate dehydrogenase is the only enzyme in the citric acid cycle that is embedded in the mitochondrial membrane which makes it associated with the electron transport chain.
What citric acid cycle enzyme is also a component of the electron transport chain?
Succinate dehydrogenase is a component of Complex II.
Malonate is a competitive inhibitor of succinate dehydrogenase. How will the concentrations of citric acid cycle intermediates change immediately after the addition of malonate? Why is malonate not a substrate for succinate dehydrogenase?
Succinate will increase in concentration followed by alpha ketogluterate and the other intermediates "upstream" of the site of inhibition. Succinate has two methylene groups that are required for the dehydrogenation whereas malonate has but one.
Nucleoside
Sugar + Base
Source of energy on earth
Sun Photosynthetic organisms use light as energy to synthesize carbohydrates Non-photosynthetic organisms use these carbohydrates as energy sources
3° structure of DNA
Supercoiling
Chymotrypsinogen Where is it synthesized and stored? No of AAs? How is it activated? Which function does it serve?
Synthesized and stored in pancreas 245 amino acids Activated by trypsin cleaves AAs 14, 15 and 147, 148 The 3 remaining peptide chains are held together by disulfide bonds Gives chymotrypsin which serves as digestive proteolytic enzyme
pH
System for measuring the strength of different acids and bases pH = log[H+] in mol/l
Branching enzyme
Takes 7 unit polymer from growing branch and forms a new α-1,6 linked branch
*Prokaryotes for synthesis: Promoters, operators, are where RNA synthesis starts. Know where it ends (regions) and know how transcription ends
Termination of RNA synthesis occurs when the growing RNA molecule can form a hairpin structure from an inverted palindromic sequence, that usually consists of a GC-rich region followed by numerous U's. This hairpin causes the RNA Polymerase to pause and the weakly bound poly-U sequence allows the RNA to dissociate from the DNA template. OR there are Rho proteins that cause the RNA to dissociate from the DNA template
Know the difference between tetrahydrofolate and S-adenosylmethionine
Tetrahydrofolate - Carries a variety of 1C compounds in a variety of oxidation states S-Adenosylmethionine - Donates methyl groups and cleaves to yield adenosine and homocysteine (which regenerates it
Under some conditions mitochondrial ATP synthase has been observed to run in reverse. How would that situation affect the proton motive force?
The ATP synthase would pump protons at the expense of ATP hydrolysis thus maintaining the proton motive force. The synthase would function as an ATPase. There is some evidence that damaged mitochondria use this tactic to maintain at least temporarily the proton motive force.
How could cupric arsenite (arsenic found in paint) make you sick?
The arsenite would have inhibited enzymes that require lipioc acid, notable, they pyruvate dehydrogenase complex.
The citric acid cycle is part of aerobic respiration but no O2 is required for the cycle. Explain this paradox.
The citric acid cycle depends on a stead sypply of NAD+ which is typically generated from NADH by reaction of the NADH with oxygen. If there is no oxygen to accept the electrons the citric acid cycle will cease to operate.
What would be the likely effect of a mutation that would prevent σ from dissociating from the RNA polymerase core?
The core enzyme without σ binds more tightly to the DNA template than does the holoenzyme. The retention of σ after chain initiation would make the mutant RNA polymerase less processive. Hence, RNA synthesis would be much slower than normal.
What structural feature of mitochondria corresponds to the thylakoid membranes?
The cristae
Iron is a component of tmany of the electorn carriers of the electron transport chain. How can it participate in a series of coupled redox reactions of the delta E0 prime value is .77 V as see in table 20.1?
The delta E0' value of iron can be altered by changing the environment of the ion.
Why can't the reactions of the glycolytic pathway simply be run in reverse to synthesize glucose?
The delta G for the reverse of glycolysis is +96 Kj/mol far to endergonic to take place.
What is the difference between DNA and RNA?
The difference between DNA and RNA is centered on the ribose sugar moiety (Figure 32.3). In ribose, both the 2' and 3' carbons have hydroxyl (-OH) groups. In deoxyribose, the 3' carbon has a hydroxyl (-OH) group but the 2' carbon has a Hydrogen (-H).
Why is the regulation of phophofructokinase by energy charge not as important in the liver as it is in muscle?
The energy needs of a muscle cell vary widely from rest to intense exercise. Consequently the regulation of phosphofructokinase by energy charge is vital. In other tissues such as the liver ATP concentration is less likely to fluctuate and will not be a key regulator of phosphofructokinase.
What are two sucicide inhibitors? What do they specifically target (enzyme) ?
The enzyme targets are thymidylate synthase and dihydrofolate reductase. Two inhibitors are fluorouracil and fluorodeoxyuridylate.
How does the phosphorolation of glycogen phosphorolase occur? What is the associated signal cascade? What enzymes are involved (protein kinase a, phosphoralase kinase)and how they interact via phosphoralation and how protein phosphatase 1 turns them off.
The hormones initiate signal cascades (Figure 24.11) that are centered on cAMP. The hormone binds to a 7TM receptor on the cell surface, resulting in the formation of cAMP within the cell which then activates Protein Kinase A, which activates Phosphorylase Kinase, which in turn phosphorylates Phosphorylase b converting it into the active Phosphorylase a. If you turn on a signal cascade, you need to turn it off. This is done by specific phosphatases. Protein phosphatase 1 removes phosphoryl groups from phosphorylase kinase and glycogen phosphorylase, thereby inactivating the enzymes.
*Know how the repressor molecule is the bolder on train tracks (know how it is removed):
The lac repressor protein is constitutively produced (i.e., all the time) and in the absence of allolactose it binds to the operator, preventing RNA Polymerase from transcribing the operon (so it just sits on the operon like the boulder on train tracks). When an inducer molecule (allolactose) binds to the repressor, it changes its conformation so that it can no longer bind to the operator, clearing the way for RNA Polymerase to transcribe the operon. There is a whole catabolized activator protein system that brings in RNA polymerase: RNA Polymerase can also be recruited to promoter regions by Catabolite Activator Protein (CAP). When CAP binds cAMP (which will increase in concentration when glucose runs out), it changes its conformation so that it can bind to promoter regions and recruit RNA Polymerase. If lactose is also present, then the way is clear for transcription of the lac operon. This selectively turns on only those operons for the metabolism of available carbon substrate.
Eukaryotes: 20 pounds of DNA in 5 pound organism: linear DNA
The linear DNA is wound around proteins. -Histone proteins form an octomer (+ charge). -The negative phosphodiester backbone of the linear DNA wraps around the octomer. -Histone protein H1 helps DNA-protein octomers (nucleosomes) associate -Histone octomer protein tails stick out so they can interact with other proteins to enable transcription -DNA wrapped around histone octomers forms a left-handed superhelix
What is cholesterols main function, what is it a precursor to. What is the enzyme that catalyzes the rate limiting step of cholesterol biosysnthesis? The full name not HMG CoA reductase
The main role is membrane fluidity. The enzyme is 3-hydroxy-3-methylglutaryl CoA reductase. Cholesterol is a precursor to steroid hormones such as progesterone testosterone, estradiol and cortisol.
Fluid mosaic model
The model describes the cell membrane as a two-dimensional liquid that restricts the lateral diffusion of membrane components
Chemiosmotic coupling
The process of coupling the electron transport chain to phosphorylation of ADP at the inner mitochondrial membrane, it is based upon a proton gradient (high [H+] in intermembrane space, low [H+] in matrix)
What is beta oxidation? What are the substrates and products of one round?
The process that breaks down fatty acids. The substrates are acyl CoA dehydrogenase, trans-delta2-enol CoA hydratase,, L-3-hydroxyacyl CoA. The products are NADH FADH2, acetyl CoA and a short fatty acid chain.
How do these get out of the mitochondria, what is the rate limiting step for urea biosynthesis? What is the antiporter that gets one compound into and out of the mitochondria.
The rate limiting step is Carbamoyl phosphate synthetase. The antiporters are transaminases or amiotransferases.
What reaction in the citric acid cycle results in the direct formation of a molecule of ATP?
The reaction catalyzed succinyl CoA synthetase. Succinyl CoA + Pi + ADP -> sucinate + CoA + ATP.
The synthesis of citrate from acetyl CoA and oxaloacetate is a biosynthetic reaction. What is the energy source that drives the formation of citrate?
The reaction is powered by the hydrolysis of a thioester. Acetyl CoA provides the thioester that is converted into citryl CoA. When this thioester is hydrolyzed citrate is formed in an irreversible reaction.
Explain how light harvesting complexes enhance the efficiency of photosynthesis.
These complexes absorb more light than can a reaction center al0ne. The light harvesting complexes funnel light to the reaction centers.
Why do we have an adjunct that runs at the same time as glycolysis? (pentose phosphate pathway) What is the oxidative part and the non oxidative part of the pathway?
These pathways that are alternatives to the Glycolytic Pathway are important for the intermediates they provide. NADPH is produced in the oxidative part and Ribose 5-phosphate is produced in the nonoxidative phase
Fatty acid molecules are hydrophobic and stored in adipose tissue and broken down in the mitochondria in tissues that need them, how do they get into the cello and through the cytoplasm what makes them soluable and what are the carriers that get them into the mitochondria? Names of stuff.
They get into the cell via ketone bodies. The carriers that get them into the mitochondria are acetoacetate and beta hydroxybutyrate.
What coenzymes are required by the pyrubate dehydrogenase complex and what are their roles?
Thiamine pyrophosphate plays a role in the decarboxylation of pyruvate. Lipoic acid transfers the acetyl group. COA accepts the acetyl group from lipoic acid to form acetyl Coa. FAD accepts the electrons and hydrogen ions when oxidized lipoic acid is reduced. NAD+ accepts electrons from FADH2.
FAtty acid break down occurs in the cytoplasm and beta oxidation takes place in the mitochondria. Why?
This avoids a futile cycle.
Site of light reactions
Thylakoid (membrane)
Know what 2 enzymes are targets for anticancer therapy, and why. Know specific inhibitors (if mentioned)
Thymidylate synthase & dihydrofolate reductase -Attach suicide inhibitors so Thymidine can't be produced and be incorporated into DNA by cancer cells. Aminopterin and Methotrexate are competitive inhibitors for dihydrofolate reductase.
Adenine triphosphate structure and formation
Triphosphate: 1) Phosphoric acid + alcohol -> ester of phosphoric acid (1 P) 2) phosphoric acid ester (1 P) + phosphoric acid ester (1 P) -> phosphoric acid anhydride (2 P) 3) phosphoric acid anhydride (2 P) + phosphoric acid ester (1 P) ATP: Triphosphate-O-ribose-adenine (purine)
SDS-PAGE
Type of chromatography used to separate proteins based on MW SDS disrupts non-covalent interactions and binds to proteins, giving them a negative charge relative to their size -> equal charge-to-mass ratio ß-ME disrupts covalent interactions Stacking gel increases resolution Low % pH 6.8 High % pH 8.8 Ion sandwich: Trailing ions (glycine) Sample Leading ions (Cl-) from Tris buffer -> when passaging into separation gel, glycine ions are accelerated (negatively charged and small) squeezing ion sandwich together giving narrower bands Glycerol increases sample's density facilitating sample loading Bromophenol blue is tracking dye Western blot
Tyrosine 3- and 1-letter code
Tyr Y
Enzyme activity is given in
U μmol substrate conversion/min
Specific enzyme activity
U/mg protein Protein concentration is needed
Glycogen synthesis- know what each one does
UDP-glucose pyrophosphorylase - combines Glucose 1-phosphate and UTP -> UDP-Glucose and PPi (hydrolyzing 2 high energy bonds to activate glucose) Glycogen Synthase - adds UDP-Glucose to the non-reducing ends of glycogen molecules, releasing UDP and forming a glycogen chain Glycogen Branching Enzyme - Takes a 7 residue fragment from the non-reducing end of a glycogen molecule (11+ residues long) and forms an α 1-6 glycosidic bond at least 4 residues away. Glycogen synthase - control point of glycogen synthesis Glycogen phosphorylase - control point of glycogen degredation -Know how it is regulated covalently
ump to ctp
UMP+ATP-[nucleotide kinase]->UDP UDP+ATP-[nucleotide kinase]->UTP
UTP to CTP
UTP can be converted to CTP with help of glutamine and ATP catalyzed by CTP synthase
Fatty acids
Unbranched or branched alkyl chain (12-20) Carboxylic acid group
Effect of FA composition in membranes
Unsaturated fatty acids cause greater fluidity
Dark reactions
Use ATP and NADPH to fix carbon Calvin cycle
Lineweaver-Burk plot
Used to determine Vmax
Valine 3- and 1-letter code
Val V
Desiccation
Vaporization -> killing by drying out (viruses and endospores can resist)
Paratope
Variable portion which binds to epitope on antigen
Know where vitamin B6 fits into all of this
Vitamin B6 derives pyridoxal phosphate, which is essential for aminotransferases
Why is vitamin D considered a vitamin?
Vitamin D is derived from cholesterol Synthesis involves one step mediated by ultraviolet light therefore it is not always possible to be synthesized
Vitamin D excretion
Vitamin D is made soluble by 2 hydroxylations at 1' and 25' (1,25-Cholecalciferol)
Competitive inhibition
Vmax can be reached by increasing substrate concentration Km increases
What is cumulative feedback inhibition?
When the end product shuts down the pathway when it reaches a high enough concentration.
What are a couple of phospholipids? Phosphotidate, phosphoglycerols. How are phospholipids made?
When you do phospholipid synthesis from phosphotidate, you are going to put other things on the phosphate group, you can either activate the phosphotidate or you can activate the alcohol. When you activate things, and this goes back to glycogen biosynthesis, we usually use nucleotide-triphosphates and then condense that with whatever we are trying to polymerize or synthesize because they make good leaving groups. Be able to compare and contrast that in glycogen biosynthesis as well as phospholipid biosynthesis. It's the same thing: we are making a good leaving group by using the nucleotide.
Why might the genomic analyses of dogs be particularly useful for investigating the genes responsible for body size and other physical characteristics?-
Within a single species, individual dogs show enormous variation in body size and substantial diversity in other physical characteristics. Therefore, genomic analyses of individual dogs would provide valuable clues concerning the genes responsible for the diversity within the species.
Disk recovery / Adaptation
[Ca2+] decreases until threshold Guanylyl cyclase is activated and inhibits PDE -> [cGMP] increases -> opens cation channels again Arrestin dissociates, Rhodopsin is dephosphorylated -> all-trans-Retinal is replaced by 11-cis-Retinal Rhodopsin kinase (RK) phosphorylates Rh with 11-cis-Retinal + Arrestin binds Rhodopsin inactivating it for another cycle of excitation
What is Phosphotidate?
a central intermediate in lipid metabolism
FA synthesis intermediates and enzymes
acetoacyl-ACP [ß-ketoacyl-ACP reductase] D-ß-hydroxybutyryl-ACP [ß-hydroxyacyl-ACP dehydratase] Crotonyl-ACP [2,3-trans-Enoyl-ACP reductase]
*Amino acid metabolism: If you are going to degrade an amino acid, you have to get rid of the amide group (first step):
amino group is transferred to α-ketoglutarate to form glutamate, which is then deaminated to form ammonia (NH4+) *amino acid + α-ketoglutaratenitrogenless amino acid +glutamate *glutamate + NAD+ + H2Oα-ketoglutarate + NADH + H+ + NH4+
What are bile salts, what type do we produce, how are they excreted, what are they made from, how are they stored, what do they do?
bile salts (detergents that solubilize dietary lipids): synthesized in the liver, stored in the gall bladder. They are made from cholesterol. They help to bring excess lipids to the intestines for excretion. They themselves aren't excreted they are recycled and used again.
How does cAMP activate protein kinase?
by binding to its regulatory subunits they catalyze the phosphorylation of next kinase or target protein
Glycolipids
carbohydrate which is linked covalently to a lipid via a glycosidic linkage
*Know about glutamate dehydrogenase
catalyzes reaction of glutamate -> ammonia. reforming α-ketoglutarate and also reduces NAD+ into NADH. Found in the Mitochondria. Helps sequester the free ammonia that is very toxic. GDP and ADP activate glutamate dehydrogenase, while GTP and ATP inhibit glutamate dehydrogenase.
Second messengers
e.g. cAMP PIP2 and Ca2+
Glucocerebroside
e.g. Glucose + Ceramide
Receptor Tyrosine kinases Class II
e.g. INSULIN receptor Dimer per subunit one cysteine-rich domain tyrosine kinase domains on inside
Receptor Tyrosine kinases Class III
e.g. fibroblast growth factor FGF receptor Monomer Immunoglobulin-like domains instead of Cys-rich region
Phosphoacylglycerols
e.g.: Phosphatidyl ester Phosphatidic ester (Phospholipids)
*Know about the urea cycle
enables us to excrete excess nitrogen. Urea cycle occurs in the liver, converting NH4+ into urea to be excreted. REGULATION: by N-acetylglutamate
FGF receptor
fibroblast growth factor binds to receptor tyrosine kinase class III
ribulose-5-phosphate
formed in pentose phosphate pathway used for purine and pyrimidine biosynthesis
Definition reduction
gaining a hydrogen ion and an electron
Types of biomolecules
glycerol and 3 fatty acids -> lipids amino acids -> proteins nucleotides -> nucleic acids monosaccharides -> polysaccharides
Nucleosome
histone + DNA
lysyl or prolyl hydroxylase
hydroxylates lys and pro needs vitamin c (ascorbic acid) as cofactor collagen is held together by H-bonds between hydroxylysine and hydroxyproline
Fibrous proteins
insoluble in water polypeptide chains are organized approximately in parallel mechanically strong e.g. keratin or collagen in connective tissue
4° structure of DNA
interaction of DNA with histones H1 H2A H2B H3 H4
Key enzymes glyoxylate pathway
isocitrate lyase malate synthase
Anomers
isomers that differ at a new asymmetric carbon atom formed on ring closure α- and β-glucose
Definition oxidation
losing a hydrogen ion and its electron
Enzymes
lower activation energy for (catalyze) a reaction affect kinetics but NOT thermodynamics (Thermodynamics state how the equilibrium is but not how fast it is reached. Kinetics states how fast equilibrium is reached)
[Anabolism] CAC main intermediates involved in gluconeogenesis and lipid anabolism
malate into PEP via PEP carboxykinase Citrate into Acetyl-CoA and Oxaloacetate via ATP-citrate lyase Acetyl CoA goes into FA synthesis
Enzyme activity
moles of substrate conversion per time [mol/s]
We test E.coli AP activity for
pH dependency Km and Vmax of pNPP mode of inhibition of free phosphate (feedback inhibition) decrease in activity in presence of a chelating agent (removing Zn ions from substrate)
pNPP
para-Nitrophenolphosphate (artificial substrate for AP) pNPP -AP-> pNP + Pi para-Nitrophenol strongly absorbs at 405 nm
Column chromatography separation based upon
polarity (RP-HPLC, NP-HPLC) charge (IEC) size (SEC) biospecificity (affinity chromatography) -> for identification AND separation
Hydrogen bond What is acceptor and what donor?
positive end (DONOR) of dipole is a HYDROGEN bonded to an atom of high electronegativity (ACCEPTOR; N or O) Non-covalent strong dipole-dipole interaction Mediates higher bp, mp and MW
cholesterol
precursor of steroid hormones and bile acids
Anaplerotic reaction
reaction that replenishes a citric acid cycle intermediate needed if intermediate is taken out for synthesis
Breakdown of ketogenic amino acids
results in acetyl-CoA or acetoacetyl-CoA from which ketone bodies can be formed
Breakdown of glucogenic amino acids
results in oxaloacetate or pyruvate which can be used for gluconeogenesis
Dialysis used compounds
separation of K3[Fe(CN)6] (Amax at 405 nm) from Coomassie Brilliant Blue (Amax at 620 nm)
Globular proteins
soluble in water -> polar residues outside -> unpolar residues turne inside in hydrophobic interaction involve α-barrels and β-sheets
Know how statins work
statins, example is Lovastatin, are drugs that inhibit 3-Hydroxy-3-MethylGlutaryl CoA Reductase and are used to lower serum cholesterol levels by preventing new cholesterol synthesis
Glycoside
sugar molecule at which anomeric carbon bound hydroxy group is esterified with any kind of residue
Kinds of RNA
tRNA - transfer rRNA - ribosomal mRNA - messenger snRNA - mRNA processing siRNA - artificial RNA to study gene expression miRNA - natural RNA regulating gene expression
In lipid biosynthesis there are some diseases, know what they are
tay sachs disease: inability to degrade gangliosides, this leads to neurons becoming clogged with lysosomes full of lipids (death by age 3) Respiratory Distress Syndrome: in premature infants: problems with the phospholipid important in preventing lung collapse during exhalation
Know about thiamine and its centrality in cell division (can't have DNA without it)
thymine is a product of DNA degradation and is salvaged by first being incorporated into a nucleoside by thymidine phosphorylase. Thymidine kinase then generates the nucleotide.
purine biosynthesis regulation
tightly regulated because high energy costs and complex pathways feedback inhibition
What do you pee your excess nitrogen out as what about birds and fish.
urea is humans, ammonia is fish, uric acid is birds.
***Know why some fatty acids are essential
when we don't have the correct desaturases to introduce double bonds into fatty acids where we need them, we must consume the fatty acids in our diets
Vitamin A synthesis
ß-carotene cleavage - Retinol conversion by Retinol dehydrogenase gives 11-trans-Retinal conversion by Retinal isomerase gives 11-trans-retinal
Carbohydrates of beans
α-D-Glucose (mono) Sucrose (di) Raffinose (tri) Stachyose (tetra)
Starch cleaving enzymes
α-amylase - ENDOglycosidase - hydrolysis of α-1,4 linkages ß-amylase - EXOglycosidase - hydrolysis of α-1,4 linkages Debranching enzyme - hydrolysis α-1,6-linkages at branches
2° structure
α-barrels and β-sheets
Deoxynucleotide
Phosphate + Sugar + Base
Sources of FAs for oxidation
Phosphoacylglycerols (by phospholipases) Triacylglycerol (by lipases)
Membrane animal versus plant versus prokaryotes
Plant membrane: More unsaturated fatty acids -> more fluid Animal membrane Cholesterol is characteristic -> more rigid Prokaryotes Possess other kinds of steroids
Vasopressin
Plays a role in the control of blood pressure by regulating contraction of smooth muscle; stimulates reabsorption of water by the kidneys (antidiuretic effect), thus increasing blood pressure
Monoclonal vs polyclonal antibodies
Polyclonal antibodies are not identical bind to the same antigen but DIFFERENT EPITOPE Monoclonal antibodies are identical and bind to SAME EPITOPE
Starch
Polymers of D-glucose Amylose Linear polymer 4000 D-glucose units α-1,4 Can form a complex with iodine I2 Amylopectin Highly branched polymer 24-30 D-glucose units α-1,4 linkages for linear chains α-1,6 linkages for braches
Epitope
Portion of antigen, paratope of AB binds to
PKC
Protein kinase C
Proton binding of Hemoglobin
Protons bind to AA residues of Hb - contributing to the Bohr effect
1° structure of DNA
Sequence of nucleotides
Needed in colorimetric assays:
Standard curve
Diastereomers
Stereoisomers that NO mirror images of each other
Control points in glycolysis
Targets to allosteric metabolic control: Hexokinase Phosphofructokinase Pyruvate kinase
What is the advantage of having an extensive set of thylakoid membranes in the chloroplasts?
The light reactions take place on thylakoid membranes. Increasing the membrane surface increases the number of ATP and NADH generating sites.
Know what FA synthase is, what it does
The multienzyme system that catalyzes the formation of saturated fatty acids.
What reaction serves to link glycolysis and the citric acid cycle and what is the enzyme that catalyzes the reaction?
The pyruvated dehydrogenase complex
What step in the citric acid cycle requires a molecule of inorganic phosphate?
The reaction catalyzed by succinly CoA synthtase.
Gluconeogensis takes place during intense exercise, which seems counter intuitive. Why would an organism synthesize glucose and at the same time use glucose to generate energy?
The synthesis of glucose during intense exercise provides a good example of inter-organ cooperation in higher organisms. When muscle is actively contracting lactate is produced from glucose by glycolysis. The lactate is released into the blood and absorbed by the liver where it is converted by gluconeogensis into glucose. The newly synthesized glucose is thne released and taken up by the muscle for energy generation.
AAs to be phosphorylated
Those that possess a hydroxy group Tyr Thr Ser
Threonine 3- and 1-letter code
Thr T
Steroids
Three 6-membered rings One 5-membered ring
Enables the synthesis of a wide range of AAs
Transamination ASAT - aspartate aminotransferase
Primary active transport
Transport is linked directly to hydrolysis of ATP
Phosphatidic ester
Triacylglycerol but one fatty acid is replaced by an PHOSPHORIC ACID
Liberation of fatty acids from triacylglycerols
Triacylglycerol lipase -> Diacylglycerol + FA Diacylglycerol lipase -> Monoacylglycerol + FA Monoacylglycerol lipase -> Glycerol + FA
Storage form of fatty acids
Triacylglycerols
What drug treats protozoan infections?
Trimethoprim, it binds tightly to protozoal dihydrofolate reductases but not to mammalian versions of the enzyme.
Tryptophan 3- and 1-letter code
Trp W
4° structure
Two or more subunits Dimers, trimers, tetramers...
Know about diabetes
Type 1 - Lack of β cells or low production of insulin by β cells (stuck in gluconeogenic/ketogenic modes) Type 2 - Overconsumption of carbohydrates - Eventually insulin receptors become unresponsive due to overstimulation
Small peptides acting as hormones
Vasopressin Oxytocin
Lipid soluble vitamins
Vitamin A Vitamin D Vitamin E Vitamin K
Non competitive inhibition
Vmax decreases Km unaffected
Know why some amino acids are essential
We lack the genes for the enzymes in their (usually more complex) biosynthetic pathways
Detection of antigenic properties after SDS-PAGE
Western blot
ΔS
change in entropy
ΔH
change of heat of a reaction at constant pressure
Passive transport
driven by concentration gradient 1) simple diffusion 2) facilitated diffusion channel protein
Specific activity
enzyme activity per mg of protein [mol/s*mg] Gives information about purity of enzyme
Triacylglycerols
esterification of glycerol with 3 fatty acids
How is glycogen phosphorolase regulated allolserically and covalently, which tissue and which form has significant T to R transitions.
high concentrations of AMP, which allosterically promotes the R State. Covalently they are effected by changing from the T to the R state.
end products of purine catabolism in different animals
human, dalmatian dogs, primates, birds, some reptiles Uric acid other animals: allantoin fish: allantoate
*Know where the branched chain amino acids are usually metabolized:
muscles use the branched chain amino acids (leucine, valine, and isoleucine) as energy sources during prolonged exercise and fasting. Nitrogen removed from amino acids in muscles are transported to the liver for Urea cycle processing
loss of H atoms
oxidation
Henderson Hasselbalch Equation
pH=pKa+log([b]/[a])
Calculating pI of amino acid
pI = 0,5(pKa1+pKa2) 3 ionizable groups: Take those pKas which surround the AA at 0 charge
Nitrogen fixation
reduction of N2 to NH3 Bacteria and azobacter and some cyanobacteria If they die the nitrogen becomes available as NH3