biochem e2l4 enzyme mechanism (starts q8))

what type of inhibitor?

what type of inhibitor?

competitive v. uncompetitive v. mixed inhibitors:

1. what do they do

1. competitive = compete w substrate in substrate binding site

   1. doesn’t change V max

2. uncompetitive = doesn’t compete in binding site but binds ES complex

   1. no matter how much substrate you add you can’t overcome it

3. mixed = not competitive, don’t just bind ES complex

   1. can bind ES complex and to the Enzyme w/o substrate

   2. cannot overcome with additional substrate

   3. affects both Km and Max

1. irreversible inhibitor:

2. suicide inhibitor

3. how medically relevant?

1. modify or destroy a catalytic functional group of an active site by covalent or extremely tight non covalent modification

   1. potent poisons

2. type of irreversible inhibitor that binds like a substrate and is then acted upon by enzyme

   1. at some point during catalysis converted into an irreversible inhibitor intermediate

3. suicide inhibitors medically important bc specific and become activated only when bound to enzyme (lower side effects)

1. is serine ionizable (acid/base reactions) under physiological conditions?

2. pH affect on enzyme activity

1. an AA reside that may participate in reaction catalyzed by pepsin (stomach protein is)

   1. serine pka 13 or Glu pka 3?

1. normally no bc of pka but it is in enzyme reaction

2. enzyme activity pH dependent

3. glu pka 3 close to where it operates at

define a and a’

1. a = inhibitor binding to free enzyme → EI complex

   1. competitive

2. a’ = inhibitor binding to ES complex → ESI

   1. uncompetitive or mixed

serine proteases:

1. what are they

2. what is the prototypical one

1. catalyze the hydrolytic cleavage of peptide bonds

2. chymotrypsin

chymotrypsin:

1. structure

2. catalytic triad?

3. substrate binding pocket?

4. highest level protein structure?

5. chymotrypsinogen?

1. 3 polypeptides (a, b, c chains) linked by disulfide bonds.

2. critical active site AA residues for catalytic reaction

   1. His Ser Asp

3. hydrophobic = binds (Trp/Phe/Tyr) to position substrate for active site

4. zymogen (proenzyme) form before cleavage makes it chymotripsin

   1. 
      

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2. acylation then deacylation

chymotrypsin:

1. what does it do? 3 AAs?

2. what reaction does it catalyze

1. 2 step mechanism

1. cleaves other proteins AFTER large hydrophobic/aromatic side chains (Trp/Phe/Tyr)

2. catalyzes peptide hydrolysis

3. 2 steps

   1. acylation then deacylation

1. 3 steps how did scientists discover chymotrypsin had two step mech?

2. how did they identify active site residues

1. this protease cleaves peptide bonds but also slowly cleaves esters

2. product of ester cleavage reaction (p-nitrophenol) is yellow, used yellow pigment to track the reactions progress

3. saw a curve with both a pre-steady state burst phase in addition to a steady state phase, suggested it was a 2 step reaction

1. chemically labeled the specific residues in the active site

1. chymotrypsin 2 step mechanism?

2. what happens at each step?

3. what is water’s role?

1. fast step = acylation and release of p-NP

   1. yellow product fast

2. slow step = water hydrolysis of acyl-enzyme intermediate (deacylation)

   1. peptide bond breaking step

3. water directly attacks an acyl bond in an intermediate

   1. water doesn’t attack the peptide bond of the substrate

what is histidine’s role

catalyses 2 acid base reactions

how is chymotrypsin reaction is a great example of 3 things:

1. general acid-base catalysis

2. transition state stabilization

3. covalent catalysis

involved in 2 acid-base reactions

4 parts chymotrypsin active site:

1. triad

2. oxyanion hole

3. glysine -

4. hydrophobic pocket

1. what are the jobs of Asp102 and His57

1. triad = Asp102, His57, Ser195

2. oxyanion hole

3. glysine = helps w/ hydrogen bonding

   1. reaction cleaves peptide bond after aromatic group

4. hydrophobic pocket = where aromatic ring goes

1. to establish Ser195 as a strong nucleophile (O-)

chymotrypsin acylation phase:

1. starting products -(acylation)→ ending product

2. what happens (generally)

3. 4 steps

1. E + protein -(acylation)→ ES complex

2. covalent catalysis forms an ester linkage that forms acyl enzyme and the peptide bond is broken

3. 4 steps

   1. Substrate binds active site: forms ES complex

      1. His57 acts as general base and takes proton from Ser195 to establish Ser195 as a strong nucleophile (O-)

      2. His 57+ stabilized by Asp102 carboxylate

      3. H bonded chain makes Ser better n-phile

   2. Nucleophilic attack:

      1. Ser195-O- nucleophilic attack on C=O (covalent catalysis/ester linkage) to form short lived tetrahedral acyl-enzyme intermediate/transition state

      2. substrate has short lived O- charge stabilized by H bonding w/ Ser195 and Gly193

         1. H bond only occurs in this intermediate (TS stabilization)

   3. removal of LG:

      1. tetrahedral intermediate collapses reforming C=O and breaking peptide bond

      2. His57 acts as an acid protonates N terminus of LG peptide so it can leave

      3. product 1 (c-terminal half) is released

   4. End:

      1. acyl-enzyme remains (2nd transition state) = N terminal peptide is covalently bound to Ser195 via ester linkage

      2. C term of substrate diffuses away

acid-base catalysis of His57 in acylation phase v deacylation phase

1. acylation

   1. base to make See195 a good nucleophile (O-)

   2. acid to protonate N on the LG 1 (C term half of substrate)

1. deacylation

   1. base to make H20 good nucleophile (OH-0

   2. acid to protonate Ser195 to release LG2 (N term half of substrate)

1. ES complex v acyl-enzyme intermediate

2. Gly193 role?

3. deacylation or acylation phase?

1. ES complex = double bonded O H bonded to Ser195

2. acyl-enzyme intermediate/TS= single bonded/neg charge O- stabilized by H bonding w/ Ser195 and Gly193 in the oxyanion hole

3. TS stabilization

4. acylation phase

chymotrypsin deacylation phase:

1. starting products -(acylation)→ ending product

2. what happens (generally)

3. 4 steps

1. Acyl-enzyme -(hydrolysis)→ chymotrypsin

2. ester linkage of acyl-enzyme is hydrolyzed to regenerate original chymotrypsin

4 steps

5. Substrate binds:

   1. H20 enters active site

6. Nucleophilic attack:

   1. His57 acts as base and deporotonates H20

   2. OH- nucleophilic attack on C=O (covalent catalysis/ester linkage)

   3. anion hole stabilizes short-lived tetrahedral acyl-enzyme intermediate

7. remove LG

   1. tetrahedra intermediate collapse

   2. His57 acts as an acid protonates Ser195 (-OH) facilitates displacement

   3. 2nd product (n-terminal half) diffuses

8. End:

   1. system reset, ready to accept new substrate

4 transition states

4 acid base reactions

2 covalent catalysis

differentiate b/t tetrahedral intermediate and acyl-enzyme intermediate

how are HIV proteases similar and 4 ways different than chymotrypsin?

1. similar

2. active site

3. water

4. complexes

5. cleave location

1. similar = cleaves proteases

2. active site = aspartate = aspartyl protease

3. water DOES directly attack peptide bond in HIV protease

4. no covalent enzyme-substrate complex

5. cleaves peptide bond b/t The and Pro AA pairs

HIV protease:

1. structure?

2. 3 step mechanism (difference w/ chymotrypsin)

1. homodimer (2 same proteins in quad structure), each with 2 Asp25s at the active site

2. 3 steps

   1. substate binding:

      1. hydrophobic pocket

   2. nucleophilic attack:

      1. General base catalysis: Asp25 (O-) takes H2O H+, OH- attacks carbonyl carbon

      2. tetrahedral intermediate (transition state) with C-O- stabilized by H bonding w/ Asp 25s

      3. (NO covalently attached intermediate, NO acyl-enzyme, NO oxyanion hole)

   3. remove LG:

      1. tetrahedral intermediate collapses to C=O (N term half released from 1 Asp25)

      2. other Asp25 acts as acid protonates C term half

How do Anti-HIV protease inhibitors work

1. they’re transition state analogs (mimic HIV protease’s tetrahedral transition state)

   1. OH mimics tetrahedral transition state O-

   2. benzyl gorup fits hydrophobic pocket

Enolase:

1. how does it catalyze its reaction

2. 3 parts active site

3. 2 parts reaction

1. divalent cations = uses 2 Mg2+ cofactors

2. 2 Mg2+, Lys345, Glu211

3. 2 steps

   1. Lys345 acts as general base and accepts substrate C2 proton

      1. C2 made more acidic by adjacent carboxyl (C=O)

      2. enolate intermediate C-O- charge stabilized by 2 Mg2+ (helps rearrange double bond)

   2. remove LG:

      1. Glu211 acts as general acid, protonates C-OH, and makes the -OH a better leaving group

      2. LG = H2O

1. what is antibiotic target?

2. specifically?

3. how does cross-linking happen?

   1. 2 step reaction mechanism

   2. transpeptidase?

4. why can we target peptidoglycan synthesis?

1. peptidoglycan synthesis

2. cell wall synthesis requires cross-linking of peptidoglycan molecules w/ each other

3. how

   1. peptidoglycan chains linked via D-ala containing peptidecross bridges

   2. transpeptidase carries out cross linking

4. cross linking mechanism

   1. transpeptidase active site (Ser-OH) attachés carbonyl of peptide bond of a D-ala

      1. cleaves a 2nd d-ala

      2. covalent linkage b/t substate, peptidoglycan, and transpeptidase

   2. peptide bond formation crosslinks 2 adjacent peptidoglycan chains (transpeptidase released)

      1. peptidoglycan chain #2 rather than water does 2nd nucleophilic attack

      2. transpeptidase = one peptide bond is replaced w/ another

5. because people have L-amino acids and D-ala is a D amino acid