Biochem Exam 3

Michaelis Menten equation assumptions

1. initial velocity assumption 2. equilibrium assumption 3. rate law E+S<->ES<->E+P first rxn: fwd=k1 back=k2 2nd rxn: fwd=k3 back=k4

Hb and CO2 transport

CO2 transferred by red blood cells 80% via isohydric transport (connected through level of proton concentration) and 20% via carbamino Hb Isohydric transport: see index cards

Bohr Effect

Effect of pH on oxygen affinity of hemoglobin. The higher the pH, the higher % saturation w respect to pO2. Physiological significance- saturation difference changes a lot with small pH change. Hydration of CO2 in tissues and exremities leads to proton production; these protons are taken up by Hb as oxygen dissociates. In lungs: CO2 is taken out of aqueous soln, released into air. protons taken out of soln. Dissociation constant- if you bind stonger, dissocation constant decreases

Time course of antigen elicited anitbody expresion

IgM spikes quickly- immune memory will respond within a week IgG- takes longer, typically a couple of weeks

kcat/Km; catalytic efficiency

apparent second order rate constant k3k1/(k2+k3) must be <=k1 Overall rate of a reaction=how quickly an enzyme can bind to substrate/how quickly it can do the reaction kcat/Km measurse how an enzyme performs when [S] is low- higher # means more efficient. chemical perfection of an enzyme means that time spent for enzyme/substrate to find each other>>time it takes for the enzyme/substrate t make the product and release it

Nucleophilic and electrophilic centers in enzymes

covalent catalysis results in formation of covalent bonds. enzyme components can be made into nucleophiles with removal of H

Eukaryotic chromatin modifications that ensure maintenance of organization during mitotic and meiotic cell divisions

1. ATP dependent chromatin remodeling factors twist/slide nucleosomes to expose diff areas of DNA to DNA repair, replication, TFs 2. post-translational covalent modifications of histones within nucleosome can facilitate/hinder DNA repair proteins or TFs w chromatin 3. canonical histones in nucleosome can be replaced by histone variants in DNA replication dependent deposition mechanism. Variants harbor info to respond to DNa damage conditions or override gene expression state 4. Methylation at C-5 position of cytosine residues in CpG nucleotides facilitates long term gene silencing and confers genome stability by repression of transposons and repetitive DNA elements. Achieved by recognition of methyl-cytosine by specific methyl-DNA binding proteins that recruit transcriptional repressor complexes/histone modifying activities

Dynamic conformational changes in catalytic cycle of dihydrofolate reductase

1. Dihydrofolate (substrate) binding. E:NADPH:S 2. Hydride transfer; reduces dihydrofolate. E.NADP+:P has 3 variable conformations. 3. NADP+ (cofactor) release- E:P 4. Cofactor binding: E:NADPH:P 5. Product release: E:NADPH- can go through 3 diff conformations

Catalytic mechanisms to increase enzyme effectiveness

1. Preferential binding of the transition state- accomplished by destabilization of S in ES complex, and formation of near attack conformations 2. Proximity and orientation and electrostatic effects (substrates must bind in v specific orientation- esp when two substrates @ once) 3. metal ion uses additional cofactors to carry out rxnwhich have chemical functionality that amino acids do not 4. General acid base- side chains of groups like carboxylates/lysine/histidine can contribute or accept charges/protons for rxn 5. covalent (nuc)- enzyme groups are involved covalently in rxn- bind and form covalent bonds w substrates to make intermediates

Mechanisms of DNA methylation mediated repression

1. TF does not recognize DNA w methyl, or cannot transcribe the DNA w methyl group. 2. Methyl CpG binding proteins (MBP)directly recognize methylated DNA and recruit co-repressor molecules 3. methylation brings histone deacetylases and methyl transferases- results in gene silencing 4. binding of MBPs impedes progress of RNAP

Pathways for Preinitiation complex assembly

1. Transcription factors recruit each other and eventually RNAP 2. holoenzyme pathway- similar to prokaryotic systems (sigma factor==TFIID is specificity factor, recruit rest of polymerase.) However, no specific core promoter element on eukaryotic genes is common to all genes

Transcription Attenuation Regulation of trp operon in E. Coli

1. transcription begins, transcribes stem loop trpL sequenec, which pauses polymerase 2. translation begins (technically transcription and translation are coupled). When ribosome reaches trp tRNA codons, moving ribosome releases paused polymerase. Sufficient levels of tryptophan/tryptophan charged tRNAs= Translation continues until it hits the stop codon, upon whcih translation stops insufficient levels: tRNAs are fewand ribosome will stall, which will open up 1,2 anti-anti-terminator loop which makes 3-4 antiterminator loop. This actually allows operon to be transcribed, which allows tRNA to be transcribed and then translated.

Mediator complex

20 subunit complex that links upstream regulatory sequences (enhancers) w RNA pol II and general TFs at promoter site= allows for assembly and activation of promoter Can help w Activation: mediator has 3 distinct structural domains. Tail domain interacts w transcriptional activators and links mediator w RNAP Repression: 4 subunit complex when bound to mediator, prevents its interaction w RNAP and transcription machinery. Yeast mediator in complex w RNAP: CTD marks location where c term domain of largest RNAP subnit emerges from surface of enzyme

Serine protease mechanism

3 amino acid residues involved- His-57, Asp-102, Ser-195 Asp-102 serves to orient His-57, which then acts as a general acid and base by abstracting H proton of Serine hydroxyl. Ser-195 acts as nucleophile nad forms covalent bond with peptide to be cleaved, turns sp2 C into sp3. Oxyanion intermediate stabilised by N-H's of glycine 193 and Ser 195.

Nucleosomal landscape of Yeast Gene

5' NFR and 3' NFR (directly before TSS and @ transcription termination) serve as landingpads for TFs that bring in RNAP. Higher levels of modifications (acetylation/methylation/phasing) close to TSS)

General Acid/Base catalysis

Acids=donate protons Bases=accept protons Being an acid/base has nothing to do w pH Direction of proton transfer to proton donor is indicated by the placement of the wide end of the dashed bond near the proton acceptor. Carboxylate, carbonyls are a general base and accepts H Histidine, hydroxyls are H donors.

Histone modifications in Chromatin

Aka histone code, which changes compaction lvl of chromatin. Completely compacted chomatin wraps 160bp of DNA around the histone (2 turns) These modifications are primarily done on N and C term tails of histones, which are highly charged with lysines and arginines. Also contain Serines and Threonines that can be phosphorylated. Phosphorylation occurs on H3+4; acetylation of lysines occurs on all histones, which produces peptide like bond and removes charge from lysine residue.

Globin production during human development

Alpha globin is synthesized at high levels (close to 50% of total globin synthesis) throughout postconception->through birth->48 weeks after birth beta globin is synthesized at low levels until birth, when levels start to rise towards 50% of globin synthesis Gamma globin is synthesized @ high levels until birth, where levels drop to 0 Fetal hemoglobin requires ~40 torr of O2 in order to reach 80% O2 saturation Adult Gb requires ~50 torr- higher concentrations of O2 in order to maintain oxygen saturation of blood (but not much more)

Entropic and enthalpic factors in catalysis

An enzyme active site is complementary in shape, charge, and polarity to the transition state for the reaction (to a lesser extent to substrate) enzyme substrate complementarity is the bass for specificty of enzyme catalyzed reaction This example brings two reactants together- bc position and unique binding abiity, can react w one another. enzyme binds them when proximityand orientation favor formation of transition state.

Equilibrium assumption

Assumes that free enzyme and enzyme substrate complex are at equilibrium (Ks)and only occasionally enzyme substrate complex decomposes to product and enzyme E+S<-> ES K=([E]X[S])/[ES] Assume this reaction is at eqm

Ionic binding of BPG to beta subunits of Hb

BPG has lots of (-) charge bc of all the phosphates. BPG binds in central cavity where it interacts a 2 Lys, 4 Hi, and 2 N termini (all +) charges Fetal Hb has a lower affinity for 2,3 BPG and therefore a higher affinirty for oxygen- so it can get oxygen from mother's Hb. affinities are finely tuned to atmospheric lvls of O2- fully oxygenated blood system responds in a very narrow range and can end up releasing 1/2 of its capacity of oxygen.

Activation Energy and Transition State

Barriers to chemical reactions exist bc reactant molecules must pass through a high energy transition state (activation energy) to form products. Cataylsts increase rxn rates by lowering the activation energy. Way to decrease energy barrier=adding heat- overall height of activation energy barrier decreases both ways

Enzymology Terminology

Biocatalyst=catalyst of biological origin (typically enzyme) Enzyme= macromolecule that functions as biocatalyst by increasing reaction rate. In general, enzyme catalyzes only one rxn type and operates on only a narrow range of substrates (reaction and substrate specificity). Substrate molecules are attacked @ same time (regiospecificity) and only one/preferentially one of the enantiomers of chiral substrate or of racemic mixtures is attacked Holoenzyme= enzyme cotaiing its characteristic prosthetic groups/metals Apoprotein=protein w/out its characteristic prosthetic group or metal ions. Apoenzyme also used (this enzyme generally inactive) cofactor=organic molecule or ion that is required by an enzyme for its activity. It may be attached either loosely (coenzyme) or tightly (prosthetic group) Prosthetic group=tightly bound, specific nonpolypeptide unit in a protein determining and involved in its biological activity coenzyme=low mw, non protein organic compound (often nt) participating in enzymatic rxns as dissociable acceptor or donor of chemical groups or electrions.

Transcriptional regulation in Eukaryotes

Chromatin remodeling complexes use the energy of ATP hydrolysis to move nucleosomes out of the way for transcription initiation- participate in other functions C terminal domain writers, readers, and erasers all participate in phosphorylation/detection/removal of phosphates on seriines in the genes. Tend to phosphorylate Serine 5 near initiation, but near termination site tends to phosphorylate Serine 2 (of CTD of RNAP)

Chymotrypsin in a complex w Eglin C (inhibitor)

Chymotrypsin catalytic triad (esp serine) comes extremely close to cleaved peptide bond. Catalytic triad (serine) enolate attacks carbonyl of sisscile bond. Inhibitor binds to enzyme in a way that blocks the enzyme's active site

Reversible inhibition reactions

Competitive- inhibitor competes w substrate for active site, but is unreactive. addition of more substrate can overwhelm this type of inhibitor Uncompetitive- inhibtor binds to ES compleex and makes ESI complex that is unreactive Noncompetitive- can do both- bind to active site of enzyme or bind to ES complex. Pure if eqm constant of E+I<-> EI and ES+I<-> ESI are the same mixed if those eqm constants are different

DNA Methylation inhibits gene expression

CpG=cytosine phosphate guanosine, methylates 5' on cytosine of both strands (aka 5meC). Usually 100s of these sequences back to back, which results in permanent modifications (gene silencing). New non-methylated strands of hemimethylated DNA are methylated to maintain the pattern- stable propagation through cell division Note: all epigenetic markers are copied and maintained during S phase of cell cycle Biological function of methylation is different in prokaryotes and eukaryotes. Prokaryotes- metylation has central role in host restriction/destruction of phage DNA no evidence of this in eukaryotes

pH and activity of lysozyne

E35 must be protonated to act as general acidcatalyst in first step of mechanism (pH<6.2) D52 must be deprononated to interact w positive charged o ion (pH>3.7) optimum pH is ~5 where both protnoated E35 and D52 are abundant. Changing pH would lose ability to perform rxn hydrolysis

Enzyme inactivation- irreversible inhibitors bind to enzyme and do not allow enzyme to reactivate

Ex: Penicillin is irreversible inhibitor of enzyme glycopeptide transpeptidase (has serine residue at active site), OH of serine can attack penicillin to make penicillin-enzyme complex which inactiates enzyme Ex: Aspirin acetylates PGH2 Synthase, whch results in steric hindrance of active site (serine that attacks acyl group of aspirin is close to enzyme active site) and results in enzyme deactivation (and anti inflammatory effects.

Limited proteolysis

Ex: insulin activation: removal of regulatory segment results in oligomerization of segments. Ex: pre- pancreatic proteases (zymogens) are activated by proteolytic cleavage which completes binding of substrate binding protein. Pancreatic enzymes are released as trypsinogen, which is activated by trypsin/can self digest or activate other proteases

Glu:Asp aminotransferase

Example of double displacement busubstrate mechanism Amine from glutamate or aspartate is transferred to pyrixdoxal, which transfers the amine to oxaloacetate to make aspartate or alpha keto gluterate to make glutamate

Creatine kinase

Example of random sequential mechanism- substratesmust bind to make reactants, then products are made, then released phosphorylates creatine- multiple substrates must bind for creatine to be phosphorylated, but ultimately phosphate from ATP->creatine

Transcriptional state in Prokaryotes

Ground state is accessible. Therefore, gene regulation is generally done through repression. Ground state->repressed state

Transcriptional state in Eukaryotes

Ground state is very compacted/restrictive. Gene regulation generally done through modifying chromain to make more accessible silent state (such as x chromosome in females) <-Ground state->poised state->active state

Binding of oxygen by hemoglobin

Hb must be able to bind oxygen in the lungs, release oxygen in capillaries. Sigmoid, cooperative oxygen binding curve of Hb makes this possible. When oxygen binds, it pulls Fe into plane of heme/polyphyrin ring by 0.039 nm, causes heme ring to shift to be planar as well. entire molcule of Hb also changes conformation slightly so that affinity increases.

Hemoglobin mechanism of alloestery

Heme molecule consists of an iron molecule surrounded by 4 rings (each with an N complexing the iron). When Heme in hemoglobin, 5 position is coordinated by N of histidine, 6 position can be filled by distal histidine or O2. When O2 fills 6 coordination position, Fe 2+ -> Fe3+ When the first oxygen binds to Fe in heme of Hb, heme Fe is drawn into plane of ring- which initaites series of conformational changes that are transmitted to adjacent subunits and increases their affinity for oxygen. POSITIVE cooperativity.

Hemoglobin inside erythrocytes

Hemoglobin and myoglobin are both capable of oxygen transport and storage. Hemoglobin is tetramer(found in blood) (4 chains), myglobin is a monomer (muscle tissue) Hemoglobin O2 partial pressure vs O2 saturation curve is low @ low pO2, but high @ high pO2- ideal for picking up O2 in air and dropping off in working muscle that needs oxygen. Myoglobin O2 saturation goes from ~80% O2 sat @ working muscle O2 partial pressre to almost 100% O2 sat- would not be able to drop off much oxygen.

2,3 Biphosphoglycerate

Highly ionic/anionic molecule that binds to hemoglobin and decreases its affinity for oxygen. Without 2,3-BPG, Hb curve for % O2 saturation would be very similar to myoglobin and it would not be physiologically relevant. 2,3-BPG creates sigmoid binding curve BPG is an example of a heterotropic (diff molecule binds to enzyme @ allosteric site) (-) effector (decreases Hb affinity for substrate) homotropic=substrate binds to enzyme @ allosteric site

Stabilization of peptide hydrolysis transition state by trypsin

Histidine N lone pair abstracts proton from Serine hydroxyl, which then attacks carbonyl of the peptide bond it is cleaving. Oxyanion is formed, carbonyl carbon goes from sp2->sp3. This oxyanion is stabilized by H bonding from the single bond NH groups of backbone of trypsin proteins

Organization of exons and introns for genes for Serine proteases

Histidine, aspartic acid, and serine in consecutive exons. These genes form a 3D structure that makes substrate binding favorable. Protease has to be cut before these amino acids in order to be activated.

IgG fold found in few examples of immunoglobulin gene superfamily

Ig fold primarily found on extracellular domains,which are structurally similar but not sequentially similar.

Intermediate vs Transition states

Importance of intermediate states- an enzyme may alter the rxn pathway to one that includes one or more intermediate states that resemble the transition state but have lower free energy. In the case of a single intermediate, the activation energies for formation of the intermediate state are lower than the activation energy for the uncatalyzed reaction.

Histone modifications vs Transcriptional activity (methylation of Lysine)

In active chromatin- H3 K4+K9 methylated In inactive chromatin- K9 and K27 hypermethlyated. Histone modifications dont necessarily differ, but lvl of methylation will change..Seems to be least methylation @ posted chromatin

Metal ion catlysis

Liver alcohol dehydrogenase catalyzes transfer of hydride ion from NADH to acetaldehyde to form ethanol. Zn @ active site stabilizes negative charge development of oxygen atom of acetaldehyde, leading to induced partial positive charge on the carbonyl C atom. Zn also polarizes substrate, moves e- density to oxygen atom and makes it more electronegative than normal Metal ions are usually transition metal

Nucleosome structure

Lysine side chains N terminal tails are in blue- closely associated w phosphoribose backbone of DNA Histone tail modifications (acetylation, methylation) and substitution (H3->H3.3) are all common histone differences.

Km

Michaelis constant=(k2+k3)/k1 This is a constant for a given enzyme and substrate. This is an estimate of the dissociation constant of [ES] complex [S] producing v=Vmax/2 is numerically equal to Km- the lower the Km, the better our enzyme is at working when [S] is small small Km means tight binding, high Km means weak binding.

Interface btwn TNFa and its receptors/antibody complexes

Normally TNFa binds to TNFR2, causes inflammatory response TNFa can also bind to TNFR1, which is similar but primarily binds to only one monomer. TNFa-infliximab- first generation of designed antibodies, human mouse antibody hybrid, which primarily binds to a single monomer. Not as effective as Humira TNFa-adalimumab- area that it binds is the largest and overlaps with the area that TNFRs bind, so it blocks TNFR from binding. Addition of adalimumab reduces binding of TNFR->lowered inflammatory reaction. There are many differences btwn the infliximab and adalimumab, but the antibody/antigen complex is still able to form

Salt bridges btwn different subunits inemoglobin

Occur btwn subunits but also within subunits. Salt bridges=ionic interactions- Cl- bridges + charged N term of alpha 2 and C term end of alpha1.

Rate of unimolecular reaction (A->P)

Rate is linearly dependent on [A] V=-d[A]/dt=k[A]=rate of disappearance of A However, if the reaction is catalyzed by an enzyme the rate shows saturation behavior bc enzyme has reached the limit of how much can be bound- end up w square root curve.

Operon

Set of adjacent structural genes (gene that codes for any RNA or protein product other than a regulatory factor), whose mRNA is synthesized as a single transcript. . However,all genes on an operon may not be exposed at the same level Operon includes adjacent regulatory elements that affect transcription of the structural genes. Sequence of adjacent bacterial genes all under the transcriptional control of the same operator. Largest operons are around 10-15 genes

Prokaryotic gene expression regulation (@ transcription initiation state)

Sigma subunit binds, which recruits beta,beta' subunits. Proteins can then interact with RNAP subunits. DNA wraps around RNAP; this interaction with the beta/beta' subunit brings it to the promoter site. About 8% of Ecoli genes are involved in transcription and regulation of transcription (making TFs, repressor proteins, etc)

Recruitment of Bromo-Chromo domain containing proteins by Histone modifications

Silent chromatin- H3K9 methylation recruits heterochromatin protein 1 (HP1), methylates adjacent nucleosomes, causes spreading of heterochromatin Active chromatin- H3K4 methylation recruits chromatin remodeling factor in physical association w HATs, acetylation of K residues prevents repressive modifications to occur and recruits transcription activators. Active chromatin- also can happen by K acetylation. Acetylated K's recruit chromatin remodeling complexes, which via ATPase activity displaces/twists nucleosomes to expose DNA areas for interaction w transcription machinery.

IgG antibody molecule structures

Simplest type of antibody still has 12 Ig domains. tines of fork=Fab domains (antibody binding fragments handle of fork=Fc domains flexible linkers (elbow) connect N terminal domains of Ig domains Fab fragments are floating and moving around- fragment connection is very flexible. Carbohydrate is bound in middle of Fc domains fragments of Fab domain are hypervariable, or the recognition elements that bind to antigens. Aka complimentarydetermining regions (CDRs)

Chromatin remodeling complex

Size of nucleosome would fit inside complex. Upon binding, they bind DNA and remove interactions btwn DNA and protein. Machines are ATP dependent- use te energy to loosen interactions btwn DNA and histone

steady state assumption

Steady state=state during which the enzyme substrate complex remains constant Pre-steady state= [ES] is building up quickly Michaelis Menten equation describes the reaction rate measured only when the steady state has been reached.

Regulator elements in Lac operon

These three sites are recognized and bound by regulatory subunits and sigma complex in RNAP. CRP site= cAMP receptor protein binding site, located before promoter and operator. cAMP/CRP affinity is higher when cAMP levels are high (cAMP is high when glucose is low) Promoter- includes binding sites for RNAP and lac repressor operator- repressors can bind here to repress transcription of strucutral genes. Repressor protein overlaps with where transcription is supposed to start; also does not allow RNAP to engage w -35 and -10 elements Inducer binds to repressor to decrease affinity of repressor for the operator- which results in dissociation of the inactive repressor-inducer complex from operator.

Stringent response signalling in E coli (stress response)

When cell is stressed (amino acid/carbon/other nutrient starvation), tRNAs go uncharged . When an uncharged tRNA makes its way into ribosome A site, RelA (ribosomal protein) phosphorylates GTP->alarmone->binds to RNAP and decreases its affinity for regular sigma subunits; instead increases affinity for stress related sigma subunits->stress related genes transcribed. alarmone also binds to cellular enzymes and changes activity.

Activation of Lac operon via CRP-cAMP

When glucose levels are low, cAMP levels and affinity w CRP are high. When cAMP binds to CRP, CRP is activated and able to bind DNA, and binding of this complex to DNA facilitates initiation of transcription by RNAP. Binding of CRP-cAMP complex is not sufficient to activate lac operon, as CRP only recruits RNAP. If the repressor is still attached to DNA, then RNAP is not able to bind to DNA sufficiently enough to initiate transcription low glucose->high cAMP->cAMP binds to CRP->CRP-cAMP complex binds to DNA->recruits RNAP->transcription if repressor is also gone When cAMP binds to CRP, CRP undergoes structural transition to a conformation capable of specific DNA binding in the C terminal domain.

Irreversible inhibitor: Transition state analogs

aka supercompetitive inhibitor- binds so tightlythat inhibitor inactivates enzyme Ex: proline racemase upon PYC (inhibitor) binding enzyme changes L proline->D proline Enzyme closes around inhibitor when bound- inhibitor ends up completely buried in monomer in closed form. This works because inhibitor looks like TS- which is bound way more tightly than the actual substrate, results in irreversible inhibition

Regulon

all genes regulated positively or negatively by a specific regulatory protein which binds to multiple promoters throughout the genome. Affects multiple operons simultaneously (~10s of genes)

Stimulon

all genes responding to a specific external signal by changed transcription. Can be characterized by comparing bacterial transcriptome in the presence and absence of the specific stimulus. ex: acidity responding genes. Acidity activates a # of TFs, activates # of genes, which allows bacterial to adapt to highly acidic environment.

Substrate saturation plot for an enzyme catalyzed reaction

amt of enzyme is constant, and the velocity of the rxn is determined to produce a v as a function of [S] w a shape of a rectangular hyperbola. At very high [S], v Vmax- velocity is limited only by conditions and by the amount of enzyme present (v is no longer dependent on [S]- this is called zero order kinetics

inhibition of enzyme activity

applies to situation where exogenous compounds are added that do not destroy tertiary structure but slow down overall rxn enzymes can inhibit reversibly or irreversibly Reversible inhibitors may bind @ active site or at some other site (allosteric)

initial velocity assumption

at the beginning of the reaction, there is very little product ([P] is very small), so the amount of [ES] formed from E+P reverse rxn is negligible Therefore, Michaelis Menten eqn applies to the reaction rate that is measured during the early reaction period (initial velocity) can simply reaction to E+S<->ES->E+P

Substrate binding pockets of homologous Serine proteases

binding pockets will strongly prefer the specific serine protease. Trypsin has lysine residue that extends into binding pocket- has favorable interaction w aspartate (+ and -) Chromotrypsin has phenylalanine residue extend into wide binding pocket w hydrophobic residues such as valine, isoleucine, leucine, tryptophan Elastase has alanine extend into relatively shallow/small binding pocket, which interacts w hydrophobic residues like itself

Proteases in Humans

catalyze the hydrolysis of peptide binds- the vast majority only make one or a few cuts in the proteins, which activate/deactivate proteins, or affect protein localization. ~600 active proteases in humans, categorized by their domains and catalytic mechanisms. Aspatric, cyestein, metallo, serine, and threonine proteases. Aspartic and metalloproteases use activated water molecule as nucleophile to attack scissile bond. Aspartic proteases bind the water molecule through two aspartic residues; metalloproteases bind the water molecule through Zn ion. Cysteine, Serine, Threonine use nucleophile created by catalytic triad in their active site. (H,D,S forms this triad) Proteolysis is common and irreversible (activity of proteases is regulated- proteases are synthesized as inactive pro-proteases called zymogens. Activation of these is often mediated by hydrolysis, resulting in exposure of protease active site. Activation can also occur through environmental changes (pH), but once activated, protease activity is mainly controlled by endogenous inhibitors.

General cofactors Serve as Bridges in activator dependent transcription

chromatin remodeling complexes use ATP to move nucleosomes out of the way for transcription initiation, but participate in other functions as well. Cofactors are required for transducing signals btwn gene specific activators and components of the general transcription machinery Sites for enhancers have ability to bind t specific transcription factors and proteins even when bound to nucleosomes Activators normally contain DNA binding domain contacting dspecific DNA sequences and activation domain interacting a general cofactors

Conformational selection vs induced fit

conformational selection=enzyme exists in multiple states. implies that the unbound enzyme is present in multiple conformations (which may have diff affinities for substrates), but the substrate can only bind to unbound enzyme that is in the same conformation as that of the ES complex. induced fit- implies conformational homogeneity in the unbound enzyme which is then distorted upon substrate binding

Ig fold

contains 7 or 8 beta strands that form 2 antiparallel beta sheets that form a flattened beta barrel (beta sandwich). Hydrophobic side chains (I, V, L) in center diagram illustrates topology of Ig domain- connection loops are crossing over so that the pattern of crossover remains constant.

Lactose utilization in E coli

controlled by lac operon: LacZ (beta galactosidase) hydrolyzes lactose->galactose and glucose LacY (beta galactosidase permease) allows lactose to enter the cell LacA- carries reaction that transacetylases saccharides. doesn't seem related to lactose metabolism- if this gene is damaged, the rest seem to be fine.. In the presence of an inducer, all three proteins accumulate simultaneously, but to different levels. Lactose itself leads to induction of the lactose operon, allolactase is true inducer. (minor product of beta galactosidase rn) In lab we use IPTG (induces lac opron but not cleaved by beta galactosidase- so its concentration does not change during exp.

rate constant

depends on gamma: transmission coefficient, correction btwn experiment and theory. Kb=boltzmann constant T=absolute temp in kelvin h=Plank's constant R=gas constant (8.314 J/Kmol)

Oligomerization as a method of gene expression regulation

diff subunits of the enzyme are transcribed at different levels. Ex: Lactate dehydrogenase has 4 subunits, which differ slightly based on the tissue it is located in. Muscle needs lots of pyruvate, needs NADH. Heart needs lactate as fuel- will actually oxidize lactate to pyruvate Ex: Regulatory subunit of protein kinase A (phosphorylates Serine/threonine) As cAMP concentration increases, it binds to regulatory subunits and causes them to release from catalytic subunits

Limited proteolytic fragmentation of IgG

elbow linkers btwn Fab and Fc domains are long and flexible, and therefore exposed and a good substrate for proteases. Therefore, IgG is often cleaved into 3 subunits. Each fragment is structurally more stable and not as accessible to proteases.

Example of enhancesome: ATF=2/c blablabla DNA complex

enhancesome: higher-order protein complex assembled at the enhancer and regulates expression of a target gene. For this complex- multiple proteins asssemble on a segment of DNA. Each binding of proteins induces conformational changes to allow more proteins to bind (binding is cooperative). by the end, DNA is heavily distorted.

TNFalpha-Adalimumab Fab interfaceF

extensice contact- uses quite a bit of the surface and bo chains. Fab fragment does not recognize a single fragment of TNFa, but actually recognizes 2 monomers of the trimer. Variety of interactions btwn Fab and TNFa- hydrophobic interactions, H bonding (assisted by H2O molecules), electrostatic interactions. Overall affinity is very high- higher than normal antibody/protein interactions bc it was designed/selected to outcompete naturally binding antibodies

Human monoclonal antibodies- Adalimumab (HUMIRA)

first fully human antibody that was mass produced as a drug. Targets TNF alpha, (tumor necrosis factor). Free and complexed TNFalpha structural variations: overall structure does not change much

Covalent intermediate in mecanism of lysozyme

fluorine analog binds to enzyme, and gets trapped/covalently bound to position 1. This is evidence that the rxn mechanism proceeds through a covalent intermediate.

Human chromatin

hella chromatin in tiny area. Gene expression regulated by making chromatin accessible. This access is regulated uring cell cycle.

Induced conformational change in hexokinase

hexokinase binds ATP and glucose to make glucose-6-phosphate. Binding of glucose to hexokinase induces a significant conformational change in the enzyme (single polypeptide chain, w 2 major domains)- allows transfer of phosphate to glucose, also excludes water (which might hydrolyze ATP)

Hypervariable loops in Immunoglobulin

hypervariable region is at the end of the fab domain- surfaces are highly complimentary (charge and structure) to antibody that it recognizes.

kcat

kcat=Vmax/[E]total, aka turnover # maximum # of substrate->product conversions per active site per unit of time (so the reaction that actually changes substrate->product)

Models of enzyme substrate interaction

lock and key- complementarity btwn substrate and enzyme- multiple substrates reacting w enzymes place them in corrrect comformation to react. induced fit- both enzyme and substrate are distorted on binding, so substrate is forced into conrofmation which approximates the transition state. This is NOT TRUE - enzyme benefits most from biding the transition state of substrates

Active site cleft of lysozyme

lysozyme=first enzyme that was crystallized. This rxn is 1-10/second- very slow, so easier to follow. natural reaction=hydrolysis of polysaccharides that form bacterial cell membrane. Therefore, substrate=polysaccharides. active site has protonated aspartate and deprotonated glutamate @ physiological pH Two proposed mechanisms: Phillips mechanism and Withers mechanism Withers mechanism is used in hydrolysis of NAG-NAM peptidoglycan

Serine proteases

many homologous proteases- have very similar amino acid sequence, and carry out same hydrolysis of the peptide bond. They differ in specificity (what the proteases bind to). Catalytic triad= His-57, Asp-102, Ser-195.

deoxyhemoglobin

methemoglobin=iron atoms are in oxidized Fe+ state. Binds to oxygen much weaker than 2+ form.

Myoglobin structure

monomeric heme protein that is composed of 8 helical segments allat angles to each other. Heme group (w Fe) is cradled in te center, contains Fe2+ that is oxidized to Fe3+ when bound to O2

Major classes of enzymes

oxidoreductase- oxidation reduction rxns (-CH3-> -CH2OH), or alcohol->carboxylic acid Transferase- transfer of functional groups (transferase, acetylase, aminotransferase) Hydrolases- hydrolysis rxns Lyases- group elim to form double bonds.. splitting or combining molecules. Prominent in metabolism Isomerases- Isomerization Ligases- Bond formation coupled w ATP hydrolysis

Serine protease mechanism intermediate stabilization

oxyanion hole facilitates formation of tetrahedral intermediates- N-H of Ser195 and Gly193 stabilize O- that is formed.

Enzymatic Rate enhancements

rate enhancement is ratio of rate constants for catalyzed and noncatalyzed rxns)-indicates how much faster the rxn occurs in presence of enzyme. Enzymes can accel rxns as much as 10^16 over noncatalyzed rates, but typical accel is 10^9 increase. Ex: Urease catalyzes hydrolysis of urea Catalyzed rate= 10^4; noncatalyzed rate=10^-10, so ratio of catalyzed/noncatalyzed is 10^14

Two component signalling systems (Histidine Kinase)

rely on transmembrane protein that has membrane bound extracellular sensor that senses external stimuli External stimuli activates internal histidine kinase->phosphorylates histidine residue ->transfers to aspartate on response regulator->triggers gene transcription activation/repression

Kinetic behavior of enzymes catalyzing bimolecular reactions

sequential or single displacement - all substrates mustbind before first product is formed. If substrates are added in an obligatory order, then mechanism is ordered sequential (otherwise random seq) ping pong/double displacement- one or more products are released before all substrates are bound

Recognition of methyl-CpG sites by methyl binding proteins

small hydrophobic interactions on surface results in binding- no distortion to protein or DNA of nucleosome. Binding occurs even on nucleosome (when wrapped around histones)- if multiple CpG's, then in general all are accessible to be bound to.

Gene expression regulation mediated by Riboswitches

small metabolites bind to 5' UTRs and can inhibit translation by producing secondary structures that hide Shine Delgarno sequences. When concentration of the small metabolite drops below its affinity for the riboswitch, gene expression is allowed. Binding of metabolite->gene repressed ex: Thiamine pyrophosphate riboswitch, highly conserved

Transition states

species that do not exist in free form (not stable in ground state) Transition state can be stabilized by intermediate stabilizers?

Structure of hemoglobin and myoglobin

structures of alpha and beta globin and myoglobin are all pretty similar

trp operon

synthesizes tryptophan; a larger operon w more genes, so more expensive for the cell to produce. Repressive ability of operator +attenuator is 800fold- doesn't take much repressor to end transcription at all (stop sequence @ beginning of second gene that stalls ribosome- can either stop transcription or allow RNAP to continue depending on environmental conditions.)

Beta galactosidase

tetrameric beta-gal cleaves lactose->galactose and glucose Lactose has 1-4 bond Allolactose has 1-6 bond (what induces lac operon) when glucose levels are high, beta gal+permease is low

Lac repressor tetramer

tetramerized by C terminal domain- a flexible linker. N and C terminal linker + DNA binding domain interactios are important for activity of lac repressor multiple sites that can bind to lac repressor- high occupancy and high level of lac repressor able to bind. In repressive state- HTH domain blocks major groove of DNA, N terminal subdomains are closer to each other, hinge helicies are formed. interactions btwn DNA-protein complex order the domains. In induced state (not repressing, such as in the presence of IPTG)- N terminal subdomains of dimer change, which causes the hinge helices in repressor to move apart. These hinge helicies are dimerized in repressive state, but movement makes them disordered; HTH motifs move out of major groove binding sites. Salt bridges form In presence of inducer (IPTG or lactose), N terminal subdomains of dimer move->hinge helicies move apart->HTH motifs move out of major groove binding sites->salt bridges form.

Zero order kinetics

velocity/rate of rxn is directly dependent on enzyme concentration

O2, CO, N2 binding to heme group

without oxygen bound, Fe is out of the heme plane. Oxygen binding pulls Fe (and His ligand along w it ) into plane of heme 0.039nm. F helix ends up moving toward ring. CO has a very strong bond w Fe- 90 degree angle w plane of ring O2 has 120 degree angle w myoglobin