Exam 2: CH6-10
Distinguish between the molecular mechanisms of competitive and noncompetitive inhibition.
A competitive inhibitor binds to the active site of an enzyme, preventing binding of the substrate. A noncompetitive in- hibitor binds at a site different from the active site, causing a conformational change, which renders the active site less able to bind substrate and convert it to product.
Why does a competitive inhibitor not change Vmax?
A competitive inhibitor blocks binding, not catalysis.
What is the function of a protein kinase?
A kinase is an enzyme that phosphorylates a protein using a high-energy phosphate, such as ATP, as the phosphate donor.
Why does a pure noncompetitive inhibitor not change the observed KM?
A noncompetitive inhibitor does not change the affinity of the enzyme for its substrate.
Describe the excision repair process in DNA, using the excision of thymine dimers as an example.
An exonuclease nicks the DNA near the site of the thymine dimers. Polymerase I acts as a nuclease and excises the incorrect nucleotides, then acts as a polymerase to incorporate the correct ones. DNA ligase seals the nick.
Suggest a reason why the cell membranes of bacteria grown at 20°C tend to have a higher proportion of unsaturated fatty acids than the membranes of bacteria of the same species grown at 37°C. In other words, the bacteria grown at 37°C have a higher proportion of saturated fatty acids in their cell membranes.
At the lower temperature, the membrane would tend to be less fluid. The presence of more unsaturated fatty acids would tend to compensate by increasing the fluidity of the membrane compared to one at the same temperature with a higher proportion of saturated fatty acids.
Suggest a reason why inorganic ions, such as K^+, Na^+, Ca^2+, and Mg^2+, do not cross biological membranes by simple diffusion.
Biological membranes are highly nonpolar environments. Charged ions tend to be excluded from such environments rather than dissolving in them, as they would have to do to pass through the membrane by simple diffusion.
Briefly describe the structure of chromatin.
Chromatin is the complex consisting of DNA and basic proteins found in eukaryotic nuclei
Supercoiling
Further coiling and twisting of DNA helix.
Explain how the pKa for histidine 57 is important to its role in the mechanism of chymotrypsin action.
Histidine 57 exists in both the protonated and unprotonated form during the chymotrypsin reaction. Its pKa of 6.0 makes this possible in the physiological pH range.
What is the importance of pyrophosphatase in the synthesis of nucleic acids?
Hydrolysis of the pyrophosphate product prevents the reversal of the reaction by removing a product.
Why was it necessary to specify that the DNA in Question 33 is double-stranded?
If the DNA were not double stranded, the requirement G=C and A=T would no longer exist.
Unsaturated fatty acids
In most unsaturated fatty acids, the cis isomer predominates; the trans isomer is rare have lower melting points than their saturated counterparts; the greater the degree of unsaturation, the lower the melting point
How can competitive and pure noncompetitive inhibition be distinguished in terms of KM?
In the case of competitive inhibition, the value of KM increases, while the value of KM remains unchanged in noncompetitive inhibition.
Distinguish between the concerted and sequential models for the behavior of allosteric enzymes.
In the concerted model, all the subunits in an allosteric enzyme are found in the same form, either the T form or the R form. They are in equilibrium, with each enzyme having a characteristic ratio of the T/R. In the sequential model, the subunits change individually from T to R.
Briefly describe the role of nucleophilic catalysis in the mechanism of the chymotrypsin reaction
In the first step of the reaction, the serine hydroxyl is the nucleophile that attacks the substrate peptide bond. In the second step, water is the nucleophile that attacks the acyl- enzyme intermediate.
Single-stranded regions of DNA are attacked by nucleases in the cell, yet portions of DNA are in a single-stranded form during the replication process. Explain.
In the replication process, the single-stranded portions of DNA are complexed to specific proteins.
Why does the American Heart Association recommend the use of canola oil or olive oil rather than coconut oil in cooking?
Less heart disease is associated with diets low in saturated fatty acids.
Two binding models have been developed to describe formation of the enzyme-substrate complex•
Lock-and-key model-substrate binds to that portion of the enzyme with a complementary shape. the shape of the substrate and the confirmation of the active site are complementary to one another Induced fit model-binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit. The enzyme undergoes conformational change upon binding to the substrate. the shape of the activation site becomes complementary to the shape of the substrate only after it binds to the enzyme
How does the structure of steroids differ from that of the other lipids discussed in this chapter?
Steroids contain a characteristic fused-ring structure, which other lipids do not
Types of mutations:
Substitution mutation: replacement of one base with another Insertion or Deletion mutation: the addition or loss of one or more bases to the DNA Silent mutation:one that has no effect on gene function
Competitive Inhibition
Substrate must compete with inhibitor for the active site → more substrate is required to reach a given reaction speed and parallel
Define supercoiling, positive supercoil, topoisomerase, and negative supercoil.
Supercoiling refers to twists in DNA over and above those of the double helix. Positive supercoiling refers to an extra twist in DNA caused by overwinding of the helix before sealing the ends to produce circular DNA. A topoisomerase is an enzyme that induces a single-strand break in supercoiled DNA, relaxes the supercoiling, and reseals the break. Negative supercoiling refers to unwinding of the double helix before sealing the ends to produce circular DNA.
What is the biggest difference between a ping-pong mechanism and either an ordered mechanism or random mechanism?
With a ping-pong mechanism, one product is released prior to the binding of the second substrate. With the other two, both substrates are bound before any product is released.
What is the difference between pure and mixed noncompetitive inhibition?
With pure noncompetitive inhibition, the binding of the in- hibitor does not change the affinity of the enzyme for sub- strate at all and vice versa; thus the KM does not change. With mixed inhibition, the substrate and inhibitor do affect each other such that the KM for the substrate is different in the presence of inhibitor.
DNA gyrase
a bacterial topoisomerase
Enzyme
a biological catalyst •with the exception of some RNAs that catalyze their own splicing, all enzymes are proteins
Nucleoside
a compound that consists of D-ribose or 2-deoxy-D-ribose covalently bonded to a nucleobase by a beta-N-glycosidic bond •Lacks phosphate group
Steroids
a group of lipids that have fused-ring structure of 3 six-membered rings, and 1 five-membered ring. •The steroid of most interest in our discussion of biological membranes is cholesterol
Lipids
a heterogeneous class of naturally occurring organic compounds classified together on the basis of common solubility properties •insoluble in water, but soluble in aprotic organic solvents including diethyl ether, chloroform, methylene chloride, and acetone
Coenzyme
a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction •metal ions-can behave as coordination compounds. (Zn2+, Fe2+) •organic compounds, many of which are vitamins or are metabolically related to vitamins.
Nucleotide
a nucleoside in which a molecule of phosphoric acid is esterified with an -OH of the monosaccharide, most commonly either the 3'-OH or the 5'-OH •Name based on parent nucleoside with a suffix "monophosphate" •Polymerization leads to nucleic acids. Linkage is repeated (3',5'-phosphodiesterbond)
Histone
a protein, particularly rich in the basic amino acids Lys and Arg; found associated with eukaryotic DNA. Five main types: H1, H2A, H2B, H3, H4
Messenger RNA, mRNA
a ribonucleic acid that carries coded genetic information from DNA to ribosomes for the synthesis of proteins •present in cells in relatively small amounts and very short-lived •single stranded •biosynthesis is directed by information encoded on DNA •a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3'end
DNA metabolism consists of
a set of enzyme catalyzed and tightly regulated processes that achieve these tasks •DNA is the Substrate that Encodes its Own Metabolisms
Transition state analog
a substance whose shape mimics that of a transition state
Absolute specificity
catalyzes the reaction of one unique substrate to a particular product
What is the basic purpose of tRNA
tRNA is bound to an amino acid and brings the correct amino acid to the growing peptide during the process of translation (protein synthesis)
How do scientists determine the KM of a substrate that is part of an ordered reaction with two substrates?
Whenever there are multiple substrates, the trick to determin- ing the KM of one of them is to run the reaction with saturat- ing concentrations of the other one.
Nucleic acid
a biopolymer containing three types of monomer units •a base derived from purine or pyrimidine (nucleobases) •a monosaccharide, either D-ribose or 2-deoxy-D-ribose •phosphoric acid •RNA (Ribonucleic Acid) •DNA (Deoxyribonucleic Acid)
Deoxyribonucleic acids, DNA
a biopolymer that consists of a backbone of alternating units of 2-deoxy-D-ribose and phosphate •the 3'-OH of one 2-deoxy-D-ribose is joined to the 5'-OH of the next 2-deoxy-D-ribose by a phosphodiesterbond
Glycolipid:
a compound in which a carbohydrate is bound to an -OH of the lipid •In most cases, sugar is either glucose or galactose •many glycolipidsare derived from ceramides •Glycolipids with complex carbohydrate moiety that contains more than 3 sugars are known as gangliosides
Reversible inhibitor:
a substance that binds to an enzyme to inhibit it, but can be released •competitive inhibitor:binds to the active (catalytic) site and blocks access to it by substrate •noncompetitive inhibitor: binds to a site other than the active site; inhibits the enzyme by changing its conformation
Irreversible inhibitor:
a substance that causes inhibition that cannot be reversed •usually involves formation or breaking of covalent bonds to or on the enzyme
Allosteric effector
a substance that modifies the behavior of an allosteric enzyme; may be an •allosteric inhibitor •allosteric activator
Double helix:
a type of 2°structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis structure based on X-Ray crystallography
Circular DNA (Prokaryotes):
a type of double-stranded DNA in which the 5'and 3'ends of each stand are joined by phosphodiesterbonds
All DNA polymerases require
a) Template-polymerization is guided by pre-formed parent strand according to Watson-Crick base-pairing rules. The requirement of a template provides a chemical rational for semiconservative DNA replication. b) Primer-a small segment with a free 3'-OH. A small segment of the new strand must be in place. DNA polymerases cannot initiate new DNA synthesis of the new strand. Some definitions related to DNA polymerase Primer terminus: the 3'-OH of the initiating strand Processivity: rate of synthesis rate of dissociation
Enzyme catalysis
an enzyme provides an alternative pathway with a lower activation energy •the transition state often has a different shape than either the substrate(s) or the product(s) •"True nature"of transition state is a chemical species that is intermediate in structure between the substrate and the product
Triacylglycerol (triglyceride)
an ester of glycerol with three fatty acids •natural soaps are prepared by boiling triglycerides (animal fats or vegetable oils) with NaOH, in a reaction called saponification (Latin, sapo, soap)
Allosteric enzyme
an oligomer whose biological activity is affected by other substances binding to it •these substances change the enzyme's activity by altering the conformation(s) of its 4°structure
DNA double helix unwinds at a specific point called
an origin of replication
Fatty acid
an unbranched-chain carboxylic acid, most commonly of 12 -20 carbons, derived from hydrolysis of animal fats, vegetable oils, or phosphodiacylglycerols of biological membranes •In the shorthand notation for fatty acids •the number of carbons and the number of double bonds in the chain are shown by two numbers, separated by a colon
Vitamins
are divided into two classes: lipid-soluble and water-soluble
Majority of fatty acids in biological systems
are found in the form of triacylglycerols •Solid ones are called fats •Liquid ones are called oils •Triacylglycerolsare the primary storage form of lipids (body fat) •Triacylglycerolsare less soluble in water than fatty acids due to the lack of charged carboxylate group •Triacylglycerolsare less dense than water: fats and oils float
Stereospecificity
catalyzes a reaction in which one stereoisomer is reacted or formed in preference to all others that might be reacted or formed •example: hydration of a cis alkene (but not its trans isomer) to give an R alcohol (but not the S alcohol)
Relative specificity
catalyzes the reaction of structurally related substrates to give structurally related products•
Mosaic
components in the membrane exist side-by-side as separate entities •the structure is that of a lipid bilayer with proteins, glycolipids, and steroids such as cholesterol embedded in it •no complexes, as for example, lipid-protein complexes, are formed
General acid-base catalysis:
depends on donation and acceptance of protons (proton transfer reactions)
Denaturation
disruption of 2°structure = dissociation of the double helix to two strands
Semiconservative replication
each daughter strand contains one template strand and one newly synthesized strand •Incorporation of isotopic label as sole nitrogen source (15NH4Cl) •Observed that 15N-DNA has a higher density than 14N-DNA, and the two can be separated by density-gradient ultracentrifugation
Aspartate transcarbamoylase (ATCase)
feedback inhibition Rate of ATCasecatalysis vs substrate concentration • Sigmoidal shape of curve describes allosteric behavior • ATCasecatalysis in presence of CTP or ATP
Rare tautomers of the bases can
hydrogen bond to the wrong base and become incorporated into DNA. It is thought that this is largely responsible for the basal error rate of DNA polymerization.
Amphipathic
in nature. both hydrophobic and hydrophilic
Fractional values
indicate complex mechanisms The order of a reaction is an experimentally determined quantity and any a priori assumptions could be (experimentally) proved wrong
Uncompetitive
inhibitor can bind to the ES complex but not to free E. Vmaxdecreases and KMdecreases
What does the rate of a reaction depends on?
its activation energy, deltaG°‡ •an enzyme provides an alternative pathway with a lower activation energy
Errors in the DNA sequence are called
mutations In E.coli, a replication mistake is made only once per 10^9-10^10 nucleotides added. Since the E.coli genome contains ~5×10^6 base pairs, this means that: ONE replication error occurs per1,000- 10,0000 times the entire genome is replicated.
Passive diffusion
of species (uncharged) across membrane dependent on concentration, presence of carrier protein
Catalytic Power
or Rate Enhancement •is the ratio of the enzyme-catalyzed rate to the uncatalyzedrate.
Secondary structure
the ordered arrangement of nucleic acid strands the double helix model of DNA 2°structure was proposed by James Watson and Francis Crick in 1953
Proofreading Activity
the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication. The "proof-reading" activity improves the accuracy of DNA polymerase by a factor of 10^2-10^3.
Primary Structure
the sequence of bases along the pentose-phosphodiesterbackbone of a DNA molecule •base sequence is read from the 5'end to the 3'end •System of notation single letter (A,G,C,U and T)
The concerted model explains
the sigmoidal effects •Higher L means higher favorability of free T form •Higher c means higher affinity between S and R form, less sigmoidal as well.
Order of reaction
the sum of the exponents in the rate equation
Tertiary structure
the three-dimensional arrangement of all atoms of a nucleic acid; commonly referred to as supercoiling
In replication of a eukaryotic chromosome
there are several origins of replication and two replication forks at each origin
At each origin of replication
there are two replication forks, points at which new polynucleotide chains are formed •There is one origin of replication and two replication forks in the circular DNA of prokaryotes
Fluid
there is lateral motion of components in the membrane; •proteins, for example, "float"in the membrane and can move along its plane
Enzymes that change linking number of DNA are called
topoisomerases •These enzymes are required for DNA unwinding and rewinding during transcription and replication
Functions
transport substances across membranes; act as receptor sites, and sites of enzyme catalysis
enzymes can increase the rate of a reaction by what factor?
up to 10^20 over an uncatalyzed reaction •some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions
the dimensions of the rate coefficient
will depend on the order of the reaction the rate always has units: concentration × time^-1, therefore the units of k should be adjusted accordingly (To find the units of a rate constant for a particular rate law, simply divide the units of rate by the units of molarity in the concentration term of the rate law)
DNA polymerase incorporates only
~1 incorrect base per 10^6-10^8added→ Template-directed synthesis works awfully well.
Comparison of A,B, and Z forms of DNA
• Both A and B-DNA are right-handed helices • Z-DNA is left handed • Z-DNA occurs in nature, usually consists of alternating purine-pyrimidine bases • Methylated cytosine found also in Z-DNA
The structure of chromatin
• Each "Bead" is a nucleosome • Nucleosome consists of: DNA wrapped around histone core • Recent research has shown that structure and spacing of nucleosomes is important in chromatin function.
Sequential model for cooperative binding of substrate to an allosteric enzyme
• R form is favored by allosteric activator • Allosteric inhibition also occurs by the induced-fit mechanism • Unique feature of Sequential Model of behavior: Negative cooperativity-Induced conformational changes that make the enzyme less likely to bind more molecules of the same type.
Mechanism of Action of Critical Amino Acids in Chymotrypsin
• Serine oxygen is nucleophile • Attacks carbonyl group of peptide bond
B6 Vitamins
• The B6 vitamins are coenzymes involved in amino group transfer from one molecule to another. • Important in amino acid biosynthesis
Some important questions to ask about enzyme mode of action:
• Which amino acid residues on an enzyme are in the active site and catalyze the reaction? • What is the spatial relationship of the essential amino acids residues in the active site? • What is the mechanism by which the essential amino acid residues catalyze the reaction? •As a model, we consider chymotrypsin, an enzyme of the digestive system that catalyzes the selective hydrolysis of peptide bonds in which the carboxyl group is contributed by Phe or Tyr
Temperature Transition in Lipid Bilayer
• With heat, membranes become more disordered; the transition temperature is higher for more rigid membranes; it is lower for less rigid membranes • Mobility of the lipid chains increases dramatically (Biochemical Connections, Butter Versus Margarine)
Levels of Nucleic Acids structure
•1°structure: the order of bases on the polynucleotide sequence; the order of bases specifies the genetic code •2°structure: the three-dimensional conformation of the polynucleotide backbone •3°structure: supercoiling •4°structure: interaction between DNA and proteins
Waxes
•A complex mixture of esters of long-chain carboxylic acids and alcohols •Found as protective coatings for plants and animals
While functioning as a stable storage of genetic information, the structure of DNA is far from static:
•A new copy of DNA is synthesizedwith high fidelity before each cell division •Errors that arise during or after DNA synthesis are constantly checked for, and repairs are made •Segments of DNA are rearranged either within a chromosome or between two DNA molecules giving offspring a novel DNA
Chymotrypsin
•A15-unit polypeptide remains bound to pi-chymotrypsin by a single disulfide bond •pi-chymotrypsin catalyzes the hydrolysis of two dipeptide fragments to give alpha-chymotrypsin •alpha-chymotrypsin consists of three polypeptide chains joined by two of the five original disulfide bonds •changes in 1°structure that accompany the change from chymotrypsinogen to alpha-chymotrypsin result in changes in 2°-and 3°structure as well. •alpha-chymotrypsin is enzymatically active because of its 2°-and 3°structure, just as chymotrypsinogen was inactive because of its 2°-and 3°structure
Base-pairing rules (coplementarity)
•A=T •G≡C •This means that the two DNA strands are complementary. If you know the sequence of one strand you can figure out the sequence of the second strand...
Purine Bases
•Adenineand guanineare found in in both RNA and DNA •Also good H-bond donors and acceptors •Adenine pKaat N1is 3.8 •Guanine pKaat N7is 2.4 •Neutral molecules at pH 7
Concerted Model allosteric activator and inhibitor
•An allosteric activator (A) binds to and stabilizes the R (active) form •An allosteric inhibitor (I) binds to and stabilizes the T (inactive) form •Effect of binding activators and inhibitors
Sphingolipids
•Backbone is not glycerol but sphingosine, a long-chain amino alcohol •Found in plants and animals •Abundant in nervous system •Bares structural similarity to phospholipids
Base Pairing
•Base pairing is complimentary •A major factor stabilizing the double helix is base pairing by hydrogen bonding between T-A and between C-G •T-A base pair comprised of 2 hydrogen bonds •G-C base pair comprised of 3 hydrogen bonds
Helical/Propeller Twists
•Bases that are exposed to minor groove contact with water •They twist in a "propeller twist"fashion •Results in: • less optimal base pair distance • More optimal base pair stacking (eliminates presence of water molecules)
Chymotrypsin Reaction TPCK
•Because Ser-195 and His-57 are required for activity, they must be close to each other in the active site •Results of x-ray crystallography show the definite arrangement of amino acids at the active site •In addition to His-57 and Ser-195, Asp-102 is also involved in catalysis at the active site •The folding of the chymotrypsin backbone, mostly in antiparallel pleated sheet array, positions the essential amino acids around the active-site pocket •The active site of chymotrypsin shows proximity of reactive a.a.
Mismatch Repair
•Both direct repair and excision repair mechanisms rely on the ability of cellular proteins to recognize inappropriate bases in DNA. •Mismatch repair replaces bases normally found in DNA and, therefore, are not recognized by the direct repair and excision repair mechanisms. •Eight possible mismatches: A-C, A-G, A-A, C-C, C-T, T-T, T-G, and G-G. •The 8 mismatches do not occur with equal frequency and are not repaired with equal efficiency.
Membrane Layers
•Both inner and outer layers of bilayer contain mixtures of lipids •Compositions on inside and outside of lipid bilayer can be different •This is what distinguishes the layers
Topoisomerases
•Class I: cut the phosphodiesterbackbone of one strand, pass the end through, and reseal •Class II: cut both strands, pass some of the remaining DNA helix between the cut strands, and reseal
DNA -The Basics
•Composed of two anti-parallel helicalstrands •The two chains differin sequence •The two strands are not held together covalently, but instead base pairing hydrogen bonds exist between the strands
Pyrimidine Bases
•Cytosine is found in both DNA and RNA •Thymine is found only in DNA •Uracil is found only in RNA •All are good H-bond donors and acceptors •Cytosine pKaat N3is 4.5 •Thymine pKaat N3is 9.5 •Neutral molecules at pH 7
Synthesis of the Leading and Lagging Strands
•DNA is synthesized from its 5'→ 3' end on bothDNA strands. •The leading strand is synthesized continuously in the 5' → 3' direction toward the replication fork •The lagging strand is synthesized semi-discontinuously (Okazaki fragments)also in the 5' → 3' direction, but away from the replication fork •The lagging strand fragments are joined by the enzyme DNA ligase, an enzyme that covalently links separate DNA strandstogether Okazaki fragments vary in length depending on cell type: Prokaroytes: 1000-2000 nucleotides Eukaryotes: 100-200 nucleotides
Nucleotide Functions
•Energy for metabolism (ATP) •Enzyme cofactors (NAD+) •Signal transduction (cAMP)
Asymmetric binding
•Enzymes can be stereospecific (Specificity where optical activity may pay a role) • Binding sites on enzymes must be asymmetric
Packing DNA into Euc. Cells and Organelles
•Eukaryotic chromosomes are made of chromatin. •Chromatin: a filamentous complex of DNA, histones, and other non-histone proteins. Chromatinundergoes dramatic structural changes during cell division. •DNA binding to the proteins in chromatinfoster the formation of solenoidalsupercoils. •Histones bind tightly to DNA to form a structure called a nucleosome.
Biological Membranes
•Every cell has a cell membrane (plasma membrane) •Eukaryotic cells also have membrane-enclosed organelles (nuclei, mitochondria...etc) •Plant membranes have a higher percentage of unsaturated fatty acids than animal membranes •The presence of cholesterol is characteristic of animal rather than plant membranes •Animal membranes are less fluid (more rigid) than plant membranes •The membranes of prokaryotes, which contain no appreciable amounts of steroids, are the most fluid
The advantage of fats over polysaccharides:
•Fatty acids carry more energy per carbon because they are more reduced •Fatty acids carry less water along because they are nonpolar
Information Transfer in Cells
•Information encoded in the nucleotide sequence of DNA is transcribed through RNA synthesis •Sequence then dictated by DNA sequence •Central dogma of biology
Denaturation of DNA
•It is a reversible process: Double stranded DNA can be re-formed with slow cooling (annealing)
Two types of allosteric enzyme systems exist
•K system:an enzyme for which an inhibitor or activators alters K0.5 •V system:an enzyme for which an inhibitor or activator alters Vmax but not K0.5 Note: for an allosteric enzyme, the substrate concentration at one-half Vmaxis called the K0.5
Effect of Double Bonds on the Conformations of Fatty Acids
•Kink(s) in hydrocarbon chain •Causes disorderin packing against other chains •This disorder causes greater fluidityin membranes with cis-double bonds vs...... saturated FA chains
Lewis acid/base reactions•
•Lewis acid:an electron pair acceptor •Lewis base:an electron pair donor •Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes (metal ion catalysts) •carboxypeptidase A requires Zn2+ for activity
Proteins Can be Anchored to Membranes
•N-myristoyl-and S-palmitoyl anchoring motifs •Anchors can be via N-terminal gly •Thioester linkage with Cys
Replication of DNA
•Naturally occurring DNA exists in single-stranded and double-stranded forms, both of which can exist in linear and circular forms •Difficult to generalize about all cases of DNA replication
Nucleotides and Nucleosides
•Nucleotide = •Nitrogeneousbase •Pentose •Phosphate •Nucleoside = •Nitrogeneousbase •Pentose •Nucleobase= •Nitrogeneousbase
DNA in the cell must be organized to allow
•Packing of large DNA molecules within the cells •Access of proteins to read the information in DNA sequence •There are several levels of organization, one of which is the supercoiling of the double-stranded DNA helix
Cholesterol reduces Fluidity
•Presence of cholesterol reduces fluidity by stabilizing extended chain conformations of hydrocarbon tails of FA •Due to hydrophobic interactions
Z-form is derivative of B-form
•Produced by flipping one side of the backbone 180˚ without disturbing the backbone covalent bonds or hydrogen bonds
Super DNA Coiled Topology-3oStructure
•Prokaryotic DNA is circular. It can form supercoils. • Double helix can be considered to a 2-stranded, right handed coiled rope •Can undergo positive/negative supercoiling
Pyridoxal Phosphate
•Pyridoxaland pyridoxamine phosphates are involved in the transfer of amino groups in a reaction called transamination
The Concerted Model ,The enzyme has two conformations
•R (relaxed):binds substrate tightly; the active form •T (tight or taut):binds substrate less tightly; the inactive form •in the absence of substrate, most enzyme molecules are in the T (inactive) form •the presence of substrate shifts the equilibrium from the T (inactive) form to the R (active) form •in changing from T to R and vice versa, all subunits change conformation simultaneously; all changes are concerted
The primase reaction
•RNA serves as a primer in DNA replication •primer activity first observed in-vivo .•Primase -catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence
Termination
•Replication sequences direct termination for replication. •The termination utilization substance protein (TUS) binds to these sequences and prevents the replisome from further unwinding DNA. •This facilitates the termination of replication.
Soaps
•Soaps form water-insoluble salts when used in water containing Ca(II), Mg(II), and Fe(III) ions (hard water) •Reactions with acids/bases as catalysts •Salts formed by saponification
Solubility and Melting Point of Saturated Fatty Acids
•Solubility decreasesas the chain length increases •Melting point increases as the chain length increases •Melting point decreases with unsaturation
Direct Repair
•Some damaged (or modified) nucleotides and mismatched bases are recognized and repaired by proteins that continually scan DNA in order to detect specific lesions. •These proteins effect repair without cleaving the DNA or excising the base. examples: O^6-Methylguanine-DNA methyltransferase; repairs alkylated DNA
Nucleic Acid Functions
•Storage of genetic info (DNA) •Transmission of genetic info (mRNA) •Processing of genetic information (ribozymes) •Protein synthesis (tRNAand rRNA)
In an enzyme-catalyzed reaction:
•Substrate, S = the reactant •Active site:the small portion of the enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions E + S --> ES enzyme-substrate complex
Nucleic Acids: DNA & RNA
•The genetic information of the cell is composed of DNA. •DNA is composed of two"STRANDS". •Strand is a linear sequence of (mono)nucleotides covalently linked together. •RNA is composed of one"STRAND" •Both DNA and RNA are linear (not branched) •Covalent bonds formed via phosphodiesterlinkages •Directionality: •We read the sequence for 5' to 3'
DNA is a Very Large Macromolecule
•The linear dimensions of DNA are much bigger than the virions or cells that contain them •Bacteriophages T2 and T4 are about 0.2microm long and 0.1microm wide •Fully extended T4 DNA double helix is about 60microm long •DNA in the virion or cell is organized into compact forms, typically via coiling and association with proteins
Factors Affecting DNA Denaturation
•The midpoint of melting (Tm) depends on base composition: high CG increases Tm •Tmdepends on DNA length: longer DNA has higher Tm(important for short DNA) •Tmdepends on pH and ionic strength •High salt increases Tm
Conformation of Fatty Acids
•The saturated chain tends to adopt extended conformations •The double bonds in natural unsaturated fatty acids are commonly in cis configuration •This introduces a kink in the chain Unsaturated fatty acids make less extensive favorable interactions → It takes less thermal energy to disrupt disordered packing of unsaturated fatty acids → this explains why unsaturated cis fatty acids have a lower melting point
Chymotrypsin catalyzes
•The selective hydrolysis of peptide bonds where the carboxyl is contributed by Phe and Tyr It also catalyzes hydrolysis of the ester bonds
Control of Enzyme Activity via Phosphorylation
•The side chain -OH groups of Ser, Thr, and Tyr can form phosphate esters •Phosphorylation by ATP can convert an inactive precursor into an active enzyme •Membrane transport is a common example
Summary of Replication
•Three stages: Initiation, Propagation and Termination •Requires DNA Polymerase: Catalyzes the addition of nucleoside triphosphate to a template starting at the 5'end; proceeds in the 5'→3' direction •No DNA polymerase is able to begin a new chain. It can only add a nucleotide on to a preexisting 3'-OH group. For this reason DNA polymerase needs an RNA primer at which it can add the first nucleotide.
The REPLISOME
•Topoismerases: remove and introduce supercoils •Helicase: a helix-destabilizing protein, promotes unwinding by binding at the replication fork •Single-stranded binding (SSB) protein: stabilizes single-stranded regions by binding tightly to them. •Primase: synthesis of RNA primers •DNA ligase: covalently link together Okazaki fragments
•There are two major types of topoisomerases
•Type Itopoisomerases work by making a transient cut in one DNA strand •Type II topoisomerases work by making a transient cut in both DNA strands •The energy required for this process is supplied by the hydrolysis of ATP to ADP and Pi
Phosphoacylglycerols (Phospholipids)
•When one alcohol group of glycerol is esterified by a phosphoric acid rather than by a carboxylic acid, phosphatidic acid produced •Phosphoacylglycerols(phosphoglycerides) are the second most abundant group of naturally occurring lipids, and they are found in plant and animal membranes.
Z-DNA
•a left-handed double helix •may play a role in gene expression
A-DNA
•a right-handed helix, but thicker than B-DNA •11 base pairs per turn of the helix •has not been found in vivo
Active transport
•a substance is moved against a concentration gradient •primary active transport: transport is linked to the hydrolysis of ATP or other high-energy molecule; for example, the Na+/K+ion pump •secondary active transport: driven by H+gradient
Challenges in duplication of circular double-stranded DNA
•achievement of continuous unwinding and separation of the two DNA strands •arotectionof unwound portions from attack by nucleases that attack single-stranded DNA •synthesis of the DNA template from one 5'-> 3'strand and one 3'-> 5'strand •efficient protection from errors in replication •Eukaryotic DNA is linear (different case -same principles)
Challenges in Replication
•achievement of continuous unwinding and separation of the two DNA strands •protection of unwound portions from attack by nucleases that attack single-stranded DNA •synthesis of the DNA template from one 5'→ 3' strand and one 3' → 5' strand •efficient protection from errors in replication
•DNA polymerase function has the following requirements:
•all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP •Mg2+ •an RNA primer -a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication •DNA-Pol I: repair and patching of DNA •DNA-Pol III: responsible for the polymerization of the newly formed DNA strand •DNA-Pol II, IV, and V: proofreading and repair enzymes
The key to allosteric behavior is the existence of multiple forms for the 4°structure of the enzyme
•allosteric effector:a substance that modifies the 4°structure of an allosteric enzyme •homotropic effects:allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase •heterotropic effects:allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP
In 1969 Jenks proposed that
•an immunogen would elicit an antibody with catalytic activity if the immunogen mimicked the transition state of the reaction •the first catalytic antibody or abzyme was created in 1986 by Lerner and Schultz
Base stacking
•bases are hydrophobic and interact by hydrophobic interactions •in standard B-DNA, each base rotated by 32°compared to the next and, while this is perfect for maximum base pairing, it is not optimal for maximum overlap of bases; in addition, bases exposed to the minor groove come in contact with water •many bases adopt a propeller-twist in which base pairing distances are less optimal but base stacking is more optimal and water is eliminated from minor groove contacts
Synthesis and linking of new DNA strands
•begun by DNA polymerase III •the newly formed DNA is linked to the 3'-OH of the RNA primer •as the replication fork moves away, the RNA primer is removed by DNA polymerase I
Peripheral proteins
•bound by electrostatic interactions •can be removed by raising the ionic strength
Integral proteins
•bound tightly to the interior of the membrane •can be removed by treatment with detergents or ultrasonification •removal generally denatures them
Organization of ATCase
•catalytic unit: 6 subunits organized into 2 trimers •regulatory unit: 6 subunits organized into 3 dimers •Catalytic subunits can be separated from regulatory subunits by a compound that reacts with cysteine (p-hydroxymercuribenzoate)
Lipids:Cyclic forms
•cholesterol, steroid hormones, and bile acids
B-DNA
•considered the physiological form •a right-handed helix, diameter 11Å •10 base pairs per turn (34Å) of the helix
RNA
•consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3'-OH of one pentose and the 5'-OH of the next •the pentose unit is -D-ribose (it is 2-deoxy-D-ribose in DNA) •the pyrimidine bases are uracil and cytosine (they are thymine and cytosine in DNA) •in general, RNA is single stranded (DNA is double stranded)
Passive transport
•driven by a concentration gradient •simple diffusion: a molecule or ion moves through an opening •facilitated diffusion:a molecule or ion is carried across a membrane by a carrier/channel protein
Lipids:Open Chain forms
•fatty acids, triacylglycerols, sphingolipids, phosphoacylglycerols, glycolipids, •lipid-soluble vitamins •prostaglandins, leukotrienes, and thromboxanes
Membrane Receptors
•generally oligomeric proteins •binding of a biologically active substance to a receptor initiates an action within the cell
Hydrogen bonds are broken and base stacking is lost
•most commonly by heat denaturation (melting) •as strands separate, absorbance at 260 nm increases •increase is called hyperchromicity •midpoint of transition (melting) curve = Tm •the higher the % G-C, the higher the Tm •renaturationis possible on slow cooling
Kinetics of Chymotrypsin Reaction
•p-nitrophenyl acetate is hydrolyzed by chymotrypsin in 2 stages. •At the end of stage 1, the p-nitrophenolate ion is released. •At stage 2, acyl-enzyme intermediate is hydrolyzed and acetate (Product) is released...free enzyme is regenerated
Molecular basisof membrane structure is in lipid component(s):
•polar head groups are in contact with the aqueous environment •nonpolar tails are buried within the bilayer •the major force driving the formation of lipid bilayers is the hydrophobic effect •the arrangement of hydrocarbon tails in the interior can be rigid (if rich in saturated fatty acids) or fluid (if rich in unsaturated fatty acids)
Chymotrypsinogen
•synthesized and stored in the pancreas •a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bonds •when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give -chymotrypsin Activation of chymotrypsinogen by proteolysis
Sequential Model Main Feature
•the binding of substrate induces a conformational change from the T form to the R form •the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding •sequential model represents cooperativity
Transfer RNA, tRNA
•the smallest kind of the three RNAs •a single-stranded polynucleotide chain between 73-94 nucleotide residues •carries an amino acid at its 3'end •intramolecular hydrogen bonding occurs in tRNA
Mismatches are nearly always corrected
•to correspond to the information in the template strand. → The mismatch repair system "assumes" that the template strand contains the correct base. •This means that E. coli must discriminate between the template and newly synthesized strands. •Strand discrimination is accomplished by modifying the template strand with methyl groups. •The enzyme Dam methylase methylates the N6-position of the adenine within the (5')GATC sequence.
Consider the following in light of the con- cept of levels of structure (primary, secondary, tertiary, quaternary) as defined for proteins. (a) What level is shown by double-stranded DNA? (b) What level is shown by tRNA? (c) What level is shown by mRNA?
(a) Double-stranded DNA is usually thought of as having secondary structure, unless we consider its supercoiling (tertiary) or association with proteins (quaternary). (b) tRNA is a tertiary structure with many folds and twists in three dimensions. (c) mRNA is usually considered a primary structure, as it has little other structure.
The 3'→5' exonuclease activity of DNA polymerase
(opposite to the direction of synthesis, the "proof-reading" activity) removes a mismatched base and the polymerase activity then fills in the correct base.
Reversible inhibitor can bind:
-To the free enzyme and prevent the binding of the substrate -To the enzyme-substrate complex and prevent the reaction
Irreversible inhibitors (inactivators) react with the enzyme
-one inhibitor molecule can permanently shut off one enzyme molecule -they are often powerful toxins but also may be used as drugs
Reversible inhibitorsbind to, and can dissociate from the enzyme
-they are often structural analogs of substrates or products -they are often used as drugs to slow down a specific enzyme
Give the sequence on the opposite strand for ACGTAT, AGATCT, and ATGGTA (all read 5' S 3').
. The sequence on the opposite strand for each of the follow- ing (all read 59 S 39) is ACGTAT TGCATA AGATCT TCTAGA ATGGTA TACCAT.
How are DNA lesions produced?
.We only considered errors resulting from the incorporation of the wrong base by DNA polymerase. There are other ways to produce DNA errors?
Overall error rate of DNA synthesis
1 error per 10^9-10^10 nucleotides added
Summary of DNA Replication in Prokaryotes
1)DNA synthesis is bidirectional. 2)The direction of DNA synthesis is from the 5' end to 3' end of the newly formed strand. 3)Five DNA polymerases have been found in E. coli. 4)DNA gyraseintroduces a swivel point in advance of the movement of the replication fork. 5)Primasecatalyzes the synthesis of an RNA primer. 6)The synthesis of new strands is catalyzed by Pol III. 7)Replication sequences direct termination for replication.
DNA Polymerase
1. GeneralReaction: (dNMP)n+dNTP→(dNMP)n+1+PPi 2. The reaction catalyzed by DNA polymerase is the nucleophilic attack by the 3'-OH of the growing DNA chain at the 5'-a-phosphate of the incoming dNTP. This means that DNA synthes is occurs in the 5'→3' direction. 3.The "choice" of the dNTP (from the set of dATP, dGTP, dCTP, and dTTP) is governed by the existing template strand. 4.Therefore, DNA synthesis is template directed. •The 3'-OH group at the end of the growing DNA chain acts as a nucleophile. •The phosphorus adjacent to the sugar is attacked, and then added to the growing chain.
Enzymatic Synthesis and Degradation of DNA
1.Synthesis: DNA polymerase E. coli has 5 DNA polymerases pol I pol II pol III pol IV pol V 2.Synthesis: Reverse transcriptase 3.Degradation: Nucleases Exonucleases-degrade DNA from one end, can be direction specific Endonucleases -degrade DNA from internal sites, can be non-specific or specific
What is a K system?
A K system is an allosteric enzyme in which the binding of inhibitor alters the apparent substrate concentration needed to reach one-half Vmax, S 0.5.
What is a V system?
A V system is an allosteric enzyme in which the binding of inhibitor changes the Vmax of the enzyme but not the S0.5.
Why is a clamp loader necessary in replication?
A clamp loader is necessary because the sliding clamp of DNA polymerase is a closed circle. It would not be able to get around the DNA without an enzyme to open it up.
Explain the experiment used to determine the structure of ATCase. What happens to the activity and regulatory activities when the subunits are separated?
A mercury compound was used to separate the subunits of ATCase. When the subunits were separated, one type of subunit retained catalytic activity but was no longer allosteric and was not inhibited by CTP. The other subunit type had no AT- Case activity, but it did bind to CTP and ATP.
Concerted Model, also called The Monod-Wyman-Changeaux model
A model represented by a protein having two conformations •Active (R) form-Relaxed binds substrate tightly, Inactive (T) form-Tight (taut) binds substrate less tightly both change from T to R at the same time •Substrate binding shifts equilib. To the relaxed state. Any unbound R is removed KR<KT Ratio of dissociation constants is called c (c=Kd^R/Kd^T)
What is a replication fork? Why is it important in replication?
A replication fork is the site of formation of new DNA. The two strands of the original DNA separate, and a new strand is formed on each original strand.
Why does DNA with a high A--T content have a lower transition temperature, Tm, than DNA with a high G--C content?
A-T base pairs have two hydrogen bonds, whereas G-C base pairs have three. It takes more energy and higher tem- perature to disrupt the structure of DNA rich in G-C base pairs.
Denaturation of Large DNA Molecules is not Uniform
AT rich regions melt at a lower temperature than GC-rich regions
What is the structure of ATCase?
ATCase is made up of two different types of subunits. One of them is the catalytic subunit, and there are six of them organized into two trimers. The other is the regulatory subunit, which consists of six protein subunits organized into three dimers.
What molecule acts as a positive effector (activator) of ATCase? What molecule acts as an inhibitor?
ATP acts as a positive effector of ATCase, and CTP acts as an inhibitor.
What is the difference between ATP and dATP?
ATP is made from adenine, ribose, and three phosphates linked to the 59-hydroxyl of the ribose. dATP is the same, ex- cept that the sugar is deoxyribose.
Does the behavior of allosteric enzymes become more or less cooperative in the presence of activators?
Activators make the shape of the curve less sigmoidal.
Would you expect to find adenine-guanine or cytosine-thymine base pairs in DNA? Why?
Adenine-guanine base pairs occupy more space than is available in the interior of the double helix, whereas cytosine- thymine base pairs are too small to span the distance between the sites to which complementary bases are bonded. One would not normally expect to find such base pairs in DNA.
List the substances required for replication of DNA catalyzed by DNA polymerase.
All four deoxyribonucleoside triphosphates, template DNA, DNA polymerase, all four ribonucleoside triphosphates, pri- mase, helicase, single-strand binding protein, DNA gyrase, DNA ligase.
What features distinguish enzymes that undergo allosteric control from those that obey the Michaelis-Menten equation?
Allosteric enzymes display sigmoidal kinetics when rates are plotted versus substrate concentration. Michaelis-Menten enzymes exhibit hyperbolic kinetics. Allosteric enzymes usually have multiple subunits, and the binding of substrates or effector molecules to one subunit changes the binding behavior of the other subunits.
Amino acids that are far apart in the amino acid sequence of an enzyme can be essential for its catalytic activ- ity. What does this suggest about its active site?
Amino acids that are far apart in the amino acid sequence can be close to each other in three dimensions because of protein folding. The critical amino acids are in the active site.
What is an AG/CT step?
An AG/CT step is a small section of double-stranded DNA where one strand is 59-AG-39, and the other is 59-CT-39. The exact nature of such steps greatly influences the overall shape of a double helix.
Define omega-3 fatty acid.
An omega-3 fatty acid has a double bond at the third carbon from the methyl end.
Describe the structural features of an origin of replication.
An origin of replication consists of a bubble in the DNA. There are two places at opposite ends where new polynucleotide chains are formed
Write the structural formula for a triacylglycerol, and name the component parts.
Any combination of fatty acids is possible.
Explain how phosphorylation is involved in the function of the sodium-potassium ATPase.
As part of the mechanism, the sodium potassium ATPase has an aspartate residue that becomes phosphorylated. This phosphorylation alters the conformation of the enzyme and causes it to close on one side of the membrane and open on the other, moving ions in the process.
What is the difference between B-DNA and Z-DNA?
B-DNA is a right-handed helix with specified dimensions (10 base pairs per turn, significant differences between major and minor groove, etc.). Z-DNA is a left-handed double helix with different dimensions (12 base pairs per turn, similar major and minor grooves, etc.).
DNA polymerase
Base-pairing interactions: 1 error per 10^4-10^5 nucleotides added "Proof-reading" activity: decrease in error 10^2-10^3-fold. SUM: 1 error per 10^6-10^8 nucleotides added •DNA polymerase alone DOES NOT account for the overall rate error in DNA synthesis in vivo. Another factor of 10^2-10^4-fold is observed.
A technology called PCR is used for replicat- ing large quantities of DNA in forensic science (Chapter 13). With this technique, DNA is separated by heating with an automated system. Why is information about the DNA sequence needed to use this technique?
Because any system involving replication of DNA by DNA polymerases must have a primer to start the reaction, the primer can be RNA or DNA, but it must bind to the template strand being read. Thus, enough of the sequence must be known to create the correct prime
Why can we say that having a pure non- competitive inhibitor present is similar to just having less enzyme present?
Because the inhibitor can bind to E or to ES equally well, any time there is inhibitor present, some of the enzyme will be tied up in the EIS form, which does not lead to catalysis. For this reason it would appear that less enzyme is present.
What structural features do a sphingomyelin and a phosphatidylcholine have in common? How do the structures of these two types of lipids differ?
Both sphingomyelins and phosphatidylcholines contain phosphoric acid esterified to an amino alcohol, which must be choline in the case of a phosphatidylcholine and may be choline in the case of a sphingomyelin. They differ in the second alcohol to which phosphoric acid is esterified. In phosphatidylcholines, the second alcohol is glycerol, which has also formed ester bonds to two carboxylic acids. In sphingomyelins, the second alcohol is another amino alcohol, sphingosine, which has formed an amide bond to a fatty acid.
DNA is essential to life.
Cells (usually) have only 1 or 2 copies of genomic DNA and the genetic information inherent in the nucleotide sequence must be maintained. RNA and proteins turnover in the cell quickly. Thus, mistakes made in biosynthesizing RNA and proteins are often of little consequence. Genomic DNA does not turnover; it is REPLICATED. Consequently, replication errors are permanent and are called MUTATIONS. A MUTATION is a permanent change in the nucleotide sequence of the genome.
Mechanisms by which information is transferred in the cell is based on
Central Dogma
What is the structural relationship between vitamin D3 and cholesterol?
Cholesterol is a precursor of vitamin D3; the conversion reac- tion involves ring opening.
Chymotrypsin Reaction with a model substrate
Chymotrypsin is a serine protease •DIPF inactivates chymotrypsin by reacting with serine-195, verifying that this residue is at the active site •H57 also critical for activation of enzyme •Can be chemically labeled by TPCK
Explain why cleavage of the bond between arginine 15 and isoleucine 16 of chymotrypsinogen activates the zymogen.
Chymotrypsinogen is an inactive zymogen. It is acted upon by trypsin, which cleaves peptides at basic residues, like arginine. When trypsin cleaves between the arginine and the isoleucine, chymotrypsinogen becomes semiactive, forming pi-chymotrypsin. This molecule digests itself further, forming the active alpha-chymotrypsin. As it turns out, the alpha-amino group of the isoleucine produced by the first cleavage is near the active site of alpha-chymotrypsin and necessary for its activity.
Can enzyme inhibition be reversed in all cases?
Competitive inhibition can be overcome by adding enough substrate, but this is not true for all forms of enzyme inhibition.
What are the functions of the gyrase, primase, and ligase enzymes in DNA replication?
DNA gyrase introduces a swivel point in advance of the replication fork. Primase synthesizes the RNA primer. DNA ligase links small, newly formed strands to produce longer ones.
How can breakdown in DNA repair play a role in the development of human cancers?
DNA is constantly being damaged by environmental factors and by spontaneous mutations. If these mistakes accumulate, deleterious amino acid changes or deletions can arise. As a result, essential proteins, including those that control cell di- vision and programmed cell death, are inactive or overactive, eventually leading to cancer.
Describe the discontinuous synthesis of the lagging strand in DNA replication.
DNA is synthesized from the 59 end to the 39 end, and the new strand is antiparallel to the template strand. One of the strands is exposed from the 59 end to the 39 end as a result of unwinding. Small stretches of new DNA are synthesized, still in an antiparallel direction from the 59 end to the 39 end and are linked by DNA ligase.
Describe the role of DNA ligase in the replication process.
DNA ligase seals the nicks in newly formed DNA.
Compare and contrast the properties of the enzymes DNA polymerase I and polymerase III from E. coli.
DNA polymerase I is primarily a repair enzyme. DNA polymerase III is mainly responsible for the synthesis of new DNA.
What are the common features of all DNA polymerases?
DNA polymerases have a very common structure that is often compared to a right hand, with domains referred to as the fingers, palm, and thumb. The active site where the polymerase reaction is catalyzed lies in the crevice within the palm domain. The fingers domain acts in deoxynucleotide recognition and binding, and the thumb is responsible for DNA binding.
Why is it more important for DNA to be replicated accurately than transcribed accurately?
DNA represents the permanent copy of genetic information, whereas RNA is transient. The cell could survive production of some mutant proteins, but not DNA mutation.
A health-conscious friend asks whether eat- ing carrots is better for the eyesight or for preventing cancer. What do you tell your friend? Explain
Eating carrots is good for both.VitaminA, which is abundant in carrots, plays a role in vision. Diets that include generous amounts of vegetables are associated with a lower incidence of cancer.
How does the catalytic effectiveness of enzymes compare with that of nonenzymatic catalysts?
Enzymes are many orders of magnitude more effective as catalysts than are nonenzymatic catalysts.
Give two reasons why enzyme catalysts are 10^3 to 10^5 more effective than reactions that are catalyze for example simple, H+ and OH-.
Enzymes hold the substrates in favorable spatial positions, and they bind effectively to the transition state to stabilize it. Note that all catalysts lower the activation energy, so this is not a particular enzyme function.
Suggest a reason for carrying out enzymatic reactions in buffer solutions.
Enzymes tend to have fairly sharp pH optimum values. It is necessary to ensure that the pH of the reaction mixture stays at the optimum value. This is especially true for reactions that require or produce hydrogen ions.
How is the cooperative behavior of allosteric enzymes reflected in a plot of reaction rate against substrate concentration?
Enzymes that exhibit cooperativity do not show hyperbolic curves of rate versus substrate concentration. Their curves are sigmoidal. The level of cooperativity can be seen by the shape of the sigmoidal curve.
Enzyme Specificity
Enzymes typically convert only a select group of substrates (reactants) to a select group of products. •Enzymatic reactions typically proceed without undesired side reactions. •Specific site on/in the enzyme where substrate binds and catalysis takes place is called the active site
An enzyme catalyzes the formation of ATP from ADP and phosphate ion. What is its effect on the rate of hydrolysis of ATP to ADP and phosphate ion?
Enzymes, like all catalysts, increase the rate of the forward and reverse reaction to the same extent.
Do eukaryotes have fewer origins of replication than prokaryotes, or more origins, or the same number?
Eukaryotes usually have several origins of replication, whereas prokaryotes have only one.
Is the following statement true or false? Why? "The flow of genetic information in the cell is always DNA S RNA S protein."
False. In retroviruses, the flow of information is RNA S DNA.
Fats Provide Efficient Fuel Storage
Glucose and glycogen are for short-term energy needs, quick delivery •Fats are for long term (months) energy needs, good storage, slow delivery •Fat content of a normal human enables them to survive starvation for 2-3 months, the glycogen supply would only offer a day!
Explain how glycogen phosphorylase is controlled allosterically and by covalent modification
Glycogen phosphorylase is controlled allosterically by several molecules. In the muscle, AMP is an allosteric activator. In the liver, glucose is an allosteric inhibitor. Glycogen phosphorylase also exists in a phosphorylated form and an unphosphorylated form, with the phosphorylated form being more active.
With the concerted model, what conditions favor greater cooperativity?
Greater cooperativity is favored by having a higher ratio of the T/R form. It is also favored by having a higher dissociation constant for the substrate binding to the T form.
Allosteric
Greek allo+ steric, other shape•
Suggest a reason why heating a solution containing an enzyme markedly decreases its activity. Why is the decrease of activity frequently much less when the solution contains high concentrations of the substrate?
Heating a protein denatures it. Enzymatic activity depends on the correct three-dimensional structure of the protein. The presence of bound substrate can make the protein harder to denature.
Explain the function of histidine 57 in the mechanism of chymotrypsin.
Histidine 57 performs a series of steps involving general base catalysis followed by general acid catalysis. In the first phase, it takes a hydrogen from serine 195, acting as a general base. This is followed immediately by an acid catalysis step, in which it gives the hydrogen to the amide group of the peptide bond that is breaking. A similar scheme takes place in the second phase of the reaction.
Explain, and draw a diagram to show, how acetylation or phosphorylation could change the binding affinity between DNA and histones.
Histones are very basic proteins with many arginine and lysine residues. These residues have positively charged side chains under physiological pH. This is a source of attraction between the DNA and histones because the DNA has negatively charged phosphates: Histone-NH3^+ attracts - ^O-P-O-DNA chain. When the histones become acetylated, they lose their positive charge: Histone-NH-COCH3. They therefore have no attraction to the phosphates on the DNA. The situation is even less favorable if they are phosphorylated because now both the histone and the DNA carry negative charges.
What is a homotropic effect? What is a heterotropic effect?
Homotropic effects are allosteric interactions that occur when several identical molecules are bound to a protein. The binding of substrate molecules to different sites on an enzyme, such as the binding of aspartate to ATCase, is an example of a homotropic effect. Heterotropic effects are allosteric interactions that occur when different substances (such as inhibitor and substrate) are bound to the protein. In the ATCase reaction, inhibition by CTP and activation by ATP are both heterotropic effects.
What is the energetic driving force for the formation of phospholipid bilayers?
Hydrophobic interactions among the hydrocarbon tails are the main energetic driving force in the formation of lipid bilayers.
Does proofreading always take place by the same process in replication?
In E. coli, two different kinds of exonuclease activity are possible for DNA polymerase I, which functions as a repair enzyme.
Where do lines intersect on a Lineweaver-Burk plot showing competitive inhibition? On a Lineweaver-Burk plot showing noncompetitive inhibition?
In a Lineweaver-Burk plot for competitive inhibition, the lines intersect at the y-axis intercept, which is equal to 1/Vmax. In a Lineweaver-Burk plot for noncompetitive inhibition, the lines intersect at the x-axis intercept, which is equal to -1/KM.
Suppose that you are studying a protein involved in transporting ions in and out of cells. Would you expect to find the nonpolar residues in the interior or the exterior? Why? Would you expect to find the polar residues in the interior or the exterior? Why?
In a protein that spans a membrane, the nonpolar residues are the exterior ones; they interact with the lipids of the cell membrane. The polar residues are in the interior, lining the channel through which the ions enter and leave the cell.
What structural features do a triacylglycerol and a phosphatidyl ethanolamine have in common? How do the structures of these two types of lipids differ?
In both types of lipids, glycerol is esterified to carboxylic acids, with three such ester linkages formed in triacylglycerols and two in phosphatidyl ethanolamines. The structural difference comes in the nature of the third ester linkage to glycerol. In phosphatidyl ethanolamines, the third hydroxyl group of glycerol is esterified not to a carboxylic acid but to phosphoric acid. The phosphoric acid moiety is esterified in turn to ethanolamine.
Distinguish between the lock-and-key and induced-fit models for binding of a substrate to an enzyme.
In the lock-and-key model, the substrate fits into a comparatively rigid protein that has an active site with a well-defined shape. In the induced-fit model, the enzyme undergoes a conformational change on binding to the substrate. The active site takes shape around the substrate.
Zymogen
Inactive precursor of an enzyme where cleavage of one or more covalent bonds transforms it into the active enzyme
The polymerization reaction exhibits an error rate
Incorrect bases do not base pair properly with the template and are rejected before the phosphodiester bond is made.
Noncompetitive Inhibition Inhibitor
Inhibitor and Substrate bind at distinct sites
Enzyme Inhibition
Inhibitors are compounds that decrease the rate of an enzyme catalyzed reaction
Does the behavior of allosteric enzymes become more or less cooperative in the presence of inhibitors?
Inhibitors make the shape of the curve more sigmoidal.
Michaelis-Menten Kinetics
Initial rate of an enzyme-catalyzed reaction versus substrate concentration
Explain how DNA gyrase works.
It binds to the DNA, forming loops around itself. It then cuts both strands of DNA on one part of the loop, passes the ends across another loop, and reseals
What is the metabolic role of aspartate transcarbamoylase?
It is an enzyme used in the early stages of cytidine nucleotide synthesis.
Why is it useful to plot rate data for enzymatic reactions as a straight line rather than as a curve?
It is easier to detect deviations of individual points from a straight line than from a curve.
Explain what is meant by K0.5.
K0.5 is the substrate concentration that leads to half of the maximal velocity. This term is used with allosteric enzymes, where the term KM is not appropriate.
Generation of Errors in DNA
Mammalian cells accumulate THOUSANDS of DNA errors (often called DNA lesions) each day! Fortunately, as a consequence of DNA repair, less than ONE of the DNA lesions results in a mutation
Do DNA-polymerase enzymes also function as exonucleases?
Most DNA-polymerase enzymes also have exonuclease activity.
Primary Active transport
Movement of molecules against a gradient directly linked to hydrolysis of high-energy yielding molecule (e.g. ATP)
NAD+/NADH
Nicotinamide adenine dinucleotide (NAD+) is used in many redox reactions in biology. •Contains: 1)nicotinamide ring 2) Adenine ring 3) 2 sugar-phosphate groups •NAD+is a two-electron oxidizing agent, and is reduced to NADH •Nicotinamide ring is where reduction-oxidation occurs
List three coenzymes and their functions.
Nicotinamide adenine dinucleotide, oxidation-reduction; flavin adenine dinucleotide, oxidation-reduction; coenzyme A, acyl transfer; pyridoxal phosphate, transamination; biotin, carboxylation; lipoic acid, acyl transfer.
What is a direct way of repairing double-stranded DNA breaks?
Nonhomologous DNA end joining (NHEJ) or recombination.
Do all enzymes display kinetics that obey the Michaelis- Menten equation? Which ones do not?
Not all enzymes follow Michaelis-Menten kinetics. The kinetic behavior of allosteric enzymes does not obey the Michaelis-Menten equation.
Nucleophilic substitution catalysts-
Nucleophilic electron-rich atom attacks electron deficient atom. •same type of chemistry can occur at active site of enzyme: SN1, SN2
DNA synthesis always takes place from the 5' to the 3' end. The template strands have opposite directions. How does nature deal with this situation?
One strand of newly formed DNA uses the 39-to-59 strand as a template. The problem arises with the 59-to-39 strand. Nature deals with this issue by using short stretches of this strand for a number of chunks of newly formed DNA. They are then linked by DNA ligase
What are the three most common mechanisms for enzyme-catalyzed reactions that have two substrates?
Ordered, random, and ping-pong.
Which is more hydrophilic, cholesterol or phospholipids? Defend your answer.
Phospholipids are more hydrophilic than cholesterol. The phosphate group is charged, and the attached alcohol is charged or polar. These groups interact readily with water. Cholesterol has only a single polar group, an -OH.
What role does phosphorylation of tyrosine residues play in the action of receptor proteins?
Phosphorylation of tyrosine residues can activate or deactivate a receptor protein, depending on the specific system.
Why is a short RNA primer needed for replication?
Polymerase III does not insert a deoxyribonucleotide without checking to see that the previous base is correct. It needs a previous base to check even if that base is part of a ribonucleotide.
DNA replication is bidirectionalin most organisms
Polynucleotide chains are synthesized in both directions from the origin of replication
If circular B-DNA is positively supercoiled, will these supercoils be left- or right-handed?
Positive supercoils in circular DNA will be left-handed.
What is propeller twist?
Propeller twist is a movement of the two bases in a base pair away from being in the same plane.
Why does propeller-twist occur?
Propeller-twist reduces the strength of the hydrogen bond but moves the hydrophobic region of the base out of the aqueous environment, thus being more entropically favorable.
Do all proteins associated with membranes span the membrane from one side to another?
Proteins that are associated with membranes do not have to span the membrane. Some can be partially embedded in it, and some associate with the membrane by noncovalent interactions with its exterior.
If we made a Lineweaver-Burk plot of an irreversible inhibitor, which type of reversible inhibition would it be most likely to resemble?
Pure noncompetitive.
What was the recent change in the estimated number of Pol III enzymes that are associated with the replisome?
Recently, it was concluded that three pol III enzymes are associated with the replisome instead of two.
What is the Holliday Model?
Recombination occurs by the breakage and reunion of DNA strands so that physical exchange of DNA parts takes place. The mechanism was deduced in 1964 by Robin Holliday and is referred to as the Holliday Model.
What is homologous recombination?
Recombination that involves a reaction between homologous sequences.
Prokaryotic Replication
Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands as templates
Define replication, transcription, and translation.
Replication is the production of new DNA from a DNA template. Transcription is the production of RNA from a DNA template. Translation is the synthesis of proteins directed by mRNA, which reflects the base sequence of DNA.
Suggest a reason why it would be unlikely for replication to take place without unwinding the DNA helix.
Replication requires separating the strands of DNA. This can- not happen unless the DNA is unwound.
Why is it necessary to unwind the DNA helix in the replication process?
Separating the two strands of DNA requires unwinding the helix.
What are the two essential amino acids in the active site of chymotrypsin?
Serine and histidine are the two most critical amino acids in the active site of chymotrypsin.
What amino acids are often phosphorylated by kinases?
Serine, threonine, and tyrosine are the three most often phosphorylated amino acids in proteins that are acted upon by kinases. Aspartate is another one that is often phosphorylated.
Hydrolysis of the glycosidicBond
Slow Rate, ~10^-5day Loss of purine results in an apurinic site Loss of pyrimidine results in an apyrimidinic site
Proteins, nucleic acids, and carbohydrates are grouped by common structural features found within their group. What is the basis for grouping substances as lipids?
Solubility properties (insoluble in aqueous or polar solvents, soluble in nonpolar solvents). Some lipids are not at all structurally related.
Is it unusual that the -subunits of DNA polymerase III that form a sliding clamp along the DNA do not contain the active site for the polymerization reaction? Explain your answer.
Some enzymes have a recognition site that is not the same as the active site. In the specific case of DNA polymerase III, the sliding clamp tethers the rest of the enzyme to the template. This ensures a high degree of processivity.
How does the replication process take place on a supercoiled DNA molecule?
Specific enzymes exist to cut the DNA and give a supercoiled configuration at the replication fork that allows replication to proceed.
Which of the following statements is (are) true? (a) The two strands of DNA run parallel from their 5' to their 3' ends. (b) An adenine-thymine base pair contains three hydrogen bonds. (c) Positively charged counterions are associated with DNA. (d) DNA base pairs are always perpendicular to the helix axis.
Statement (c) is true. Statements (a) and (b) are false. Statement (d) is true for the B form of DNA but not for the A and Z forms.
Which statements are consistent with the known facts about membrane transport? (a) Active transport moves a substance from a region in which its concentration is lower to one in which its concentration is higher. (b) Transport does not involve any pores or channels in membranes. (c) Transport proteins may be involved in bringing substances into cells.
Statements (a) and (c) are correct; statement (b) is not correct because ions and larger molecules, especially polar ones, require channel proteins.
Which of the following statements is (are) consistent with what is known about membranes? (a) A membrane consists of a layer of proteins sandwiched be- tween two layers of lipids. (b) The compositions of the inner and outer lipid layers are the same in any individual membrane. (c) Membranes contain glycolipids and glycoproteins. (d) Lipid bilayers are an important component of membranes. (e) Covalent bonding takes place between lipids and proteins in most membranes.
Statements (c) and (d) are consistent with what is known about membranes. Covalent bonding between lipids and proteins [statement (e)] occurs in some anchoring motifs, but is not widespread otherwise. Proteins "float" in the lipid bilayers rather than being sandwiched between them [statement (a)]. Bulkier molecules tend to be found in the outer lipid layer [statement (b)].
Which statements are consistent with the fluid-mosaic model of membranes? (a) All membrane proteins are bound to the interior of the membrane. (b) Both proteins and lipids undergo transverse (flip-flop) diffusion from the inside to the outside of the membrane. (c) Some proteins and lipids undergo lateral diffusion along the inner or outer surface of the membrane. (d) Carbohydrates are covalently bonded to the outside of the membrane. (e) The term mosaic refers to the arrangement of the lipids alone.
Statements (c) and (d) are correct. Transverse diffusion is only rarely observed [statement (b)], and the term mosaic refers to the pattern of distribution of proteins in the lipid bilayer [statement (e)]. Peripheral proteins are also considered part of the membrane [statement (a)].
DNA differs from RNA
Sugar is 2'-deoxyribose, not ribose. • Sometimes "d"used to designate "deoxy" • Writing a DNA strand -an abbreviated notation -even more abbreviated notations d(GACAT) pdApdCpdGpdT pdACGT
Temperature dependence of catalysis
Temperature can also catalyze reaction (increase rate). Increasing temperature will eventually lead to protein denaturation
Other things being equal, what is a potential disadvantage of an enzyme having a very high affinity for its substrate?
The ES complex would be in an "energy trough," with a con- sequentially large activation energy to the transition state.
With respect to the concerted model, what is the L value? What is the c value?
The L value is the equilibrium ratio of the T/R form. The c value is the ratio of the dissociation constants for substrate and the two forms of enzyme, such that c = KR/KT.
What does the hydrolysis of GTP have to do with the actions of receptor proteins?
The action of a number of receptor proteins requires binding of GTP to a specific subunit, followed by subsequent hydrolysis.
What are some possible advantages to the cell in combining phosphorylation with allosteric control?
The allosteric effect can be faster because it is based on simple binding equilibrium. For example, if AMP is an allosteric activator of glycogen phosphorylase, the immediate increase in AMP when muscles contract can cause muscle phosphorylase to become more active and to provide energy for the contracting muscles. The phosphorylation effect requires the hormone cascade beginning with glucagon or epinephrine. There are many steps before the glycogen phosphorylase is phosphorylated, so the response time is slower. However, the cascade effect produces many more activated phosphorylase molecules, so the effects are longer and stronger.
Can the presence of a catalyst increase the amount of product obtained in a reaction?
The amount of product obtained in a reaction depends on the equilibrium constant. A catalyst does not affect that.
Under what conditions can we assume that KM indicates the binding affinity between substrate and enzyme?
The assumption that the KM is an indication of the binding affinity between the substrate and the enzyme is valid when the rate of dissociation of the enzyme-substrate complex to product and enzyme is much smaller than the rate of dissocia- tion of the complex to enzyme and substrate.
What would be the most obvious characteristic of the base distribution of a single-stranded DNA molecule?
The base distribution would not have A=T and G=C, and total purine would not be equal to total pyrimidine.
The base sequence of which type of RNA is responsible for determining the order of amino acids in a protein?
The base sequence of the mRNA.
Why does the apparent KM decrease in the presence of an uncompetitive inhibitor?
The binding of inhibitor to the ES complex to form EIS re- moves some of the ES. By LeChatelier's principle, this will tend to force the reaction to the right forming more ES. By stimulating the binding of E and S in this manner, the graph will show that the KM is reduced.
Name three proteins that are subject to the control mechanism of zymogen activation.
The digestive enzymes trypsin and chymotrypsin are classic examples of regulation by zymogens. The blood-clotting protein thrombin is another.
Explain why the second phase of the chymotrypsin mechanism is slower than the first phase.
The first phase is faster for several reasons. The serine at position 195 is a strong nucleophile for the initial nucleophilic attack. It then forms an acyl-enzyme intermediate. In the second phase, water is the nucleophile, and it takes time for water to diffuse to the right spot to perform its nucleophilic attack. It is also not as strong a nucleophile as the serine. Therefore, it takes longer for water to perform its nucleophilic attack and break the acyl-enzyme intermediate than it takes for serine to create it.
How can you recognize an enzyme that does not display Michaelis-Menten kinetics?
The graph of rate against substrate concentration is sigmoidal for an allosteric enzyme but hyperbolic for an enzyme that obeys the Michaelis-Menten equation.
Suggest a reason why animals that live in cold climates tend to have higher proportions of polyunsaturated fatty acid residues in their lipids than do animals that live in warm climates
The higher percentage of unsaturated fatty acids in membranes in cold climates is an aid to membrane fluidity.
Why does the enzyme reaction for chymotrypsin proceed in two phases?
The initial phase releases the first product and involves an acylenzyme intermediate. This step is faster than the second part, in which water comes into the active site and breaks the acylenzyme bond.
Suggest a reason for the rather large energy "overkill" in inserting a deoxyribonucleotide into a growing DNA molecule. (About 15 kcal mol-1 is used in forming a phosphate ester bond that actually requires only about a third as much energy.)
The large negative DG° ensures that the back reaction of depolymerization does not occur. Energy overkill is a common strategy when it is critically important that the process does not go in the reverse direction.
The enzyme d-amino acid oxidase has a very high turnover number because the d-amino acids are po- tentially toxic. The KM for the enzyme is in the range of 1 to 2 mM for the aromatic amino acids and in the range of 15 to 20 mM for such amino acids as serine, alanine, and the acidic amino acids. Which of these amino acids are the preferred sub- strates for the enzyme?
The low KM for the aromatic amino acids indicates that they will be oxidized preferentially.
What are some key differences in how mRNA is processed in prokaryotes versus eukaryotes?
The mRNA of eukaryotes is extensively processed compared with that of prokaryotes. The mRNA in eukaryotes is initially transcribed with numerous introns that later must be spliced out. Also, the transcripts have protections at both ends in the form of a 59 cap and a 39 poly-A tail. Neither of these occur in prokaryotic mRNA.
How do the major and minor grooves in B-DNA compare to those in A-DNA?
The major groove and minor groove in B-DNA have very different dimensions (width); those in A-DNA are much closer in width.
Nucleosides: The beta-N-GlycosidicBond
The nucleobaseis covalently linked to the 1'-carbon of the pentose, forming an N-GlycosidicBond(very stable). Glycosidicbond may adopt an antior a synconformation ➙anticonformation has the bulky portion of the base furthest from the ring ➙antiis favored and present in both B-and A-DNA
If only a few of the amino acid residues of an enzyme are involved in its catalytic activity, why does the enzyme need such a large number of amino acids?
The overall protein structure is needed to ensure the correct arrangement of amino acids in the active site.
What is the complete base composition of a double-stranded eukaryotic DNA that contains 22% guanine?
The percentage of cytosine equals that of guanine, 22%. This DNA thus has a 44% G-C content, implying a 56% A-T content. The percentage of adenine equals that of thymine, so adenine and thymine are 28% each.
One of the original structures proposed for DNA had all the phosphate groups positioned at the center of a long fiber. Give a reason why this proposal was rejected.
The phosphate groups in DNA are negatively charged at physiological pH. If they were grouped together closely, as in the center of a long fiber, the result would be considerable electrostatic repulsion. Such a structure would be unstable.
Lipid Bilayers
The polar surface of the bilayer contains charged groups •The hydrophobic tails lie in the interior of the bilayer
Does the presence of a catalyst alter the standard free energy change of a chemical reaction?
The presence of a catalyst affects the rate of a reaction. The standard free-energy change is a thermodynamic property that does not depend on the reaction rate. Consequently, the presence of the catalyst has no effect.
What effect does a catalyst have on the activation energy of a reaction?
The presence of a catalyst lowers the activation energy of a reaction.
What is the primer in DNA replication?
The primer in DNA replication is a short sequence of RNA to which the growing DNA chain is bonded.
Define processivity, and indicate the importance of this concept in DNA replication.
The processivity of a DNA polymerase is the number of nucleotides incorporated before the enzyme dissociates from the template. The higher this number, the more efficient the replication process.
What reason is enzymes much larger than synthetic catalysts
The protein fold must be sufficiently stable to form this substrate cleft and pre-organize active-site residues.
Rate can be independent of concentration of reactants
The rate may not depend on the concentration of reactants but on other factors, such as the presence of catalysts.
Comment on the dual role of the monomeric reactants in replication.
The reactants are deoxyribonucleotide triphosphates. They provide not only the moiety to be inserted (the deoxyribonucleotide) but also the energy to drive the reaction (dNTP S inserted NMP 1 PPi, PPi S 2Pi).
Show graphically how the reaction velocity depends on the enzyme concentration. Can a reaction be saturated with enzyme?
The reaction velocity remains the same with increasing en- zyme concentration. It is theoretically possible, but highly unlikely, for a reaction to be saturated with enzyme.
L Are the relative amounts of cholesterol and phophatidyl- choline the same in all the kinds of membranes found in a typical mammalian cell?
The relative amounts of cholesterol and phosphatidylcholine can vary widely in different types of membranes in the same cell
Why is the replication of DNA referred to as a semiconser- vative process? What is the experimental evidence for the semicon- servative nature of the process? What experimental results would you expect if replication of DNA were a conservative process?
The semiconservative replication of DNA means that a newly formed DNA molecule has one new strand and one strand from the original DNA. The experimental evidence for semiconservative replication comes from density- gradient centrifugation (Figure 10.3). If replication were a conservative process, the original DNA would have two heavy strands and all newly formed DNA would have light strands.
Which allosteric model can explain negative cooperativity?
The sequential model can explain negative cooperativity, because a substrate binding to the T form could induce other subunits to switch to the T form, thereby reducing binding affinity.
Define steady state, and comment on the relevance of this concept to theories of enzyme reactivity.
The steady-state assumption is that the concentration of the enzyme-substrate complex does not change appreciably over the time in which the experiment takes place. The rate of appearance of the complex is set equal to its rate of disappearance, simplifying the equations for enzyme kinetics.
In the produce department of supermarkets, vegetables and fruits (cucumbers are an example) have been coated with wax for shipping and storage. Suggest a reason why this is done.
The surface wax keeps produce fresh by preventing loss of water.
Is the term KM used with allosteric enzymes? What about competitive and noncompetitive inhibition?
The term KM should be used for enzymes that display Michaelis-Menten kinetics. Thus, it is not used with allosteric enzymes. Technically, competitive and noncompetitive inhibition are also terms that are restricted to Michaelis- Menten enzymes, although the concepts are applicable to any enzyme. An inhibitor that binds to an allosteric enzyme at the same site as the substrate is similar to a classical competitive inhibitor. One that binds at a different site is similar to a noncompetitive inhibitor, but the equations and the graphs characteristic of competitive and noncompetitive inhibition don't work the same way with an allosteric enzyme.
In lipid bilayers, there is an order-disorder transition similar to the melting of a crystal. In a lipid bilayer in which most of the fatty acids are unsaturated, would you expect this transition to occur at a higher temperature, a lower temperture, or the same temperature as it would in a lipid bilayer in which most of the fatty acids are saturated? Why?
The transition temperature is lower in a lipid bilayer with mostly unsaturated fatty acids compared with one with a high percentage of saturated fatty acids. The bilayer with the unsaturated fatty acids is already more disordered than the one with a high percentage of saturated fatty acids.
What are micro RNAs?
These are small RNAs of 20 to 22 base pairs that can bind to DNA and alter gene regulation. They are important in development and regulation of many processes.
What properties of metal ions make them useful cofactors?
They act as Lewis acids (electron-pair acceptors) and can take part in enzyme catalysis mechanisms of enzymes.
Are the sequences shown in Question 6 those of RNA or DNA? How can you tell?
They are DNA sequences because of the presence of thymine rather than uracil.
The lagging strand is synthesized in fragments, Okazaki fragments found in prokaryotes and eukaryotes
This is due to the fact that both strands are synthesized in concert by dimeric DNA polymerase situated at the replication fork. One of the 5'→3' parent strands has to wrap around one subunit so that the dimeric enzyme can move in the direction of replication fork movement despite synthesis of new DNA in the "wrong" direction, the 3'→5' direction.
You have just isolated a pure lipid that contains only sphingosine and a fatty acid. To what class of lipids does it belong?
This lipid is a ceramide, which is one kind of sphingolipid.
Would nature rely on the same enzyme to catalyze a reaction either way (forward or backward) if the deltaG° were -0.8 kcal mol^-1? If it were -5.3 kcal mol^-1?
Tothefirstquestion,most probably:local concentrations(mass- action concepts) could easily dictate the direction. To the second question, probably not: local concentrations would seldom be sufficient to overcome a relatively large DG° of -5.3 kcal in the reverse reaction.
What is the name of the process that produces RNA from a DNA template?
Transcription.
Which of the following lipids are not found in animal membranes? (a) Phosphoglycerides (b) Cholesterol (c) Triacylglycerols (d) Glycolipids (e) Sphingolipids
Triacylglycerols are not found in animal membranes.
List three proteases and their substrates.
Trypsin, chymotrypsin, and thrombin are all proteases. Trypsin cleaves peptide bonds where there are amino acids with positively charged side chains (Lys and Arg). Chymotrypsin cleaves peptides at amino acids with aromatic side chains. Thrombin cleaves the protein fibrinogen into fibrin.
How is the turnover number of an enzyme related to Vmax?
Turnover number = Vmax/[ET].
When does the kcat/KM value ap- proximate the catalytic efficiency of an enzyme?
Under conditions of low substrate concentration.
Length of fatty acid plays a role in its chemical character
Usually contain even numbers of carbons (can contain odd, depending on how they are biosynthesized) • FA that contain C=C, are unsaturated: If contain only C-C bonds, they are saturated. No conjugated bonds (for the most part)
Why are many vitamin supplements sold as antioxidants? How does this relate to material in this chapter?
Vitamins A and E are known to scavenge free radicals, which can do oxidative damage to cells.
How does proofreading take place in the process of DNA replication?
When an incorrect nucleotide is introduced into a grow- ing DNA chain as a result of mismatched base pairing, DNA polymerase acts as a 39-exonuclease, removing the incorrect nucleotide. The same enzyme then incorporates the correct nucleotide.
