Ch 9 Enzymatic Catalysis

General properties of enzymes

biocatalysts

active site

specificity

enzymes increase reaction rate

lower activation energy

x10⁶ to 10¹² rate enhancement

enzymes are classified by reaction type (7 classes)

1. oxidoreductases

2. transferases

3. hydrolases

4. Lyases

5. isomerases

6. ligases

7. translocases

oxidoreductases

oxidation-reduction reactions

e- transport reaction

transferases

transfer of functional groups

hydrolases

hydrolysis reactions

lyases

group elimination to form double bonds

isomerases

isomerization

ligases

bond formation coupled with ATP hydrolysis

translocases

movement of molecules across/within membrane

ex: flipASE

enzymes act on specific substrates

highly specific

lock and key ES model → geometric complementary and electronic complementary

induced fit→ conformational change on substrate binding, facilitates shifts of particular residues and functional groups into proper positions to catalyze reaction

Nearly all enzymes that participate in chiral reactions are

stereospecific

Some enzymes catalyze very stereospecific reactions

enzymes must act on a very stereospecific compound

Some enzymes are more permissive than others

ex: chymotrypsin mediates peptide bond hydrolysis on a wide variety of substrates and can even catalyze related reactions on different functional groups (ester bond hydrolysis)

Transition state

X^‡ = point of highest free energy

Reaction rate is proportional to

Does ∆G change with catalyst?

No, ∆G does not change with catalyst

catalysts act be lowering free energy barrier by same amount for both forward and reverse reactions

∆∆G_cat^‡ =

change in E_A

reduction in ∆G^‡ by the catalyst

Preferential transition state binding

an enzyme’s active site has greater structural complementarity to the TS than to the substrate or product itself

an optimal set of non-covalent interactions between E and S occurs specifically at transition state (ES^‡)

Transition state analogs are enzyme inhibitors

proline racemase is inhibited by planar analogs of proline

planar analogs bind to the enzyme 160x greater affinity than proline

rational basis for drug design

cofactors expand the range of enzymatic reactions

needed for specific catalyst

in order to complete the catalytic cycle, the coenzyme must return to its original state

cofactors can be metal ions or coenzymes

metal ions/metalloenzymes

common ex: Fe^2+/3+, Zn²⁺, Cu⁺, Mg²⁺

what happens when Cd²⁺ or Zn²⁺ replaces Zn²⁺

toxic!

apoenzymes

require cofactors

inactive

holoenzyme

active

apoenzyme+cofactor

coenzymes

organic molecules

many derived from vitamins

cosubstrates AND prosthetic groups

cosubstrates

transiently bound

ex: NAD+/NADH, NADP+/NADH, CoQ

prosthetic groups

permanently bound

ex: heme (includes Fe) in Mb + Hb

ex: Thiamine pyrophosphate (TPP) — aldehyde group transfer, active form of thiamine (vit. B₁)

Alcohol Dehydrogenase (ADH) Reaction

In hydrophobic environments, acids ____ pKa

increase pKa

it is more stable protonated

In hydrophobic environments, bases ___ pKa

decrease pKa

it is more stable with neutral/no charge in hydrophobic environment

When a like charge is nearby, acids ___ pKa

increase pKa

When a like charge is nearby, bases ____ pKa

decrease pKa

When there are salt bridges or H-bonds, acids ____ pK

decrease pK

When there are salt bridges or H-bonds nearby, bases ___ pK

increase pK

Proteins shift their pKa values to stabilize whatever charge state is energetically favorable in that microenvironment

1. Hydrophobic protein interior -

   1. Acids increase pKa — the group wants to remain neutral b/c hydrophobic core hates charge

   2. Bases decrease pKa — side chains positive when protonated, to escape this instability, base rapidly kicks off proton to be neutral

2. Like Charges nearby -

   1. Acids increase pKa — to prevent charge-charge repulsion, want the acid to hang on to its proton

   2. Bases Decrease pKa — positive charge next to protonated base may repel, thus base loses its proton easier

3. Salt Bridges or H-bonds -

   1. acids decrease pKa — deprotonated form is stabilized in salt bridge/H-bond, thus it is rewarded and acid gives up proton earlier

   2. bases increase pKa — protonated form if stabilized by H-bond/salt-bridge, protein rewards it for keeping its proton by base holding onto proton more

Concerted acid-base catalysis

ability of enzymes to arrange several catalytic groups around their substrates makes concerted acid-base catalysis a common mechanism

ex: Bovine pancreatic RNase A

RNase A Mechanism

RNA molecule is hydrolyzed in 2 steps

2 Histidine residues, both act as acid/base

concerted acid/base catalysis for each step

RNase A step 1

His 12 acts as a general base and His 119 acts as acid

RNase step 2

His 12 acts as a general acid and His 119 as a general base to promote Hydrolysis

Covalent (nucleophilic) catalysis

usually requires a nucleophile

transient formation of covalent bond between E & S

3 stages:

1. nucleophile reaction between catalyst and substrate to form covalent bond

2. withdrawal of e^- from reaction center by new electrophilic catalyst

3. elimination of catalyst, a reaction that is essentially reverse of step 1

biologically important nucleophiles

alkoxide (RO^-)

Thiolate (RS^-)

Neutral 1* amine (RNH₂)

Neutral imidazole

Biologically important electrophiles

Protons (H⁺)

Metal ions

Carbonyl carbon atom

Cationic imine (Schiff base)

Metal ion catalysts contributions

1. orient structures

2. redox: transfer electrons

3. electrostatics: stabilize or shield negative charges

Zn²⁺ and Carbonic Anhydrase

Zinc orients the OH group for catalysis

How do enzymes facilitate their catalysis reactions

1. enzymes bring substrates into contact with catalytic groups

2. enzymes bind their substrates in proper relative orientations for reaction

3. electrostatic catalysis - distribution of charges around catalytic site lowers free energy of T.S.

4. enzymes freeze relative translation & rotational motions of substrates

Geometry of S_N2 reaction

bimolecular, nucleophilic substitution

Lysozyme

acts on peptidoglycan

breaks B(1→4) bonds between NAM and NAG in cell wall peptidoglycan

can also break B(1→4) in chitin

found in cells/secretions

evolved to ward off gram (+) bacteria

Lysozyme cleavage site

repeating units of (GlcNAc-MurNAc)_n

HEW Lysozyme

classic antibacterial enzyme

(NAG)₆: hexamer of N-acetylglucosamine acting as an inhibitor, binds to enzyme’s groove without being easily cleaved

enzyme has a long cleft that can accommodate up to 6 residues

Glu35 and Asp52 sit right inside active site cleft — acting as acid-base pair that coordinates cleavage of carb chains

Lysozyme reaction mechanism

acid-based catalysis

• Glu acts as an acid and base

covalent catalysis — transition state binding

Lysozyme reaction mechanism step 1

binding with substrate polysaccharide

lysozyme reaction mechanism step 2

general acid base catalysis — protonate glycosidic bond

Lysozyme reaction mechanism step 3

covalent catalysis, new E-S covalent bond formation

Lysozyme reaction mechanism step 4

water binding

serine proteases

proteolytic enzyme

peculiarly reactive Ser residue

found in digestive enzymes, blood clotting, inflammatory factors, development

cleave specific for peptide bond

chymotrypsin, trypsin, elastase

chymotrypsin

serine protease cleaving for large bulky hydrophobic groups

Tryptophan, Phenylalanine, Tyrosine

trypsin

serine protease cleaving for basic, large, bulky, positive residues (arginine and lysine)

elastase

serine protease cleaving small hydrophobic residues (alanine, valine, glycine)

catalytic traid

H-bonded Ser, His, Asp residues that participate in catalysis in serine proteases and related enzymes

Specificity pocket of chymotrypsin

Specificity pocket of trypsin

cationic side chains of trypsin’s preferred residues (Arg, Lys) can therefore form ion pairs with Asp

Specificity pocket of elastase

rapidly hydrolyzes Ala, Gly, and Val-rich protein elastin

binding pocket is largely occluded by side chains of Val and Thr

active site residues in serine proteases are quite different, suggesting-

seems highly improbably they could have evolved from a common ancestor protein

evidence for convergent evolution

Serine proteases Phase 1 — Acylation

1. Nucleophilic attack & formation of 1st tetrahedral intermediate

   1. His57 acts as a base, deprotonating hydroxyl group of Ser195

   2. Ser195 becomes an alkoxide nucleophile, which attacks the carbonyl C of polypeptide backbone

2. Breakdown to Acyl-Enzyme Intermediate

   1. His57 acts as a general acid, donating proton back to N atom of peptide bond to be cleaved

   2. peptide bond breaks, splitting protein in half

3. Release of the First Product (Amine Terminus)

   1. amine product diffuses out of active site

   2. remaining half of substrate covalently attached to Ser195, forming acyl-enzyme intermediate

Serine proteases Phase 2 — Deacylation

4. Nucleophilic attack by water

   1. His57 acts as a general base, pulling proton off water molecule, making it a hydroxide ion

   2. activated water molecule attacks acyl-enzyme ester bond making 2nd tetrahedral intermediate

5. His57 acts as a general acid once more, donating a proton to break the bond between Ser195 and remaining substrate

   1. second half of peptide is released

   2. Ser195 is protonated back to its original state, resetting the enzyme so it can catalyze another reaction

TS Stabilization in Serine Proteases

• main reason activation barrier is decreased

• catalytic efficiency of serine proteases comes from preferential binding of the tetrahedral intermediate (T.S.) over initial ES complex

1. Before attack, substrate’s carbonyl is sp² hybridized and flat — sits outside of the oxyanion hole

2. Once nucleophile (Ser 195) attacks, carbon shifts to sp³ tetrahedral hybridized and a negative charge builds up on oxyanion

   1. geometric change forces oxygen atom to pop into oxyanion hole, forming 2 strong H-bonds with backbone NH of Gly 193 and Ser 195

Why does the barrier drop for chymotrypsin lowering activation energy by binding to TS state rather than ground state?

the 2 extra H-bonds only form when the substrate is forced into high-energy, tetrahedral shape

the enzyme provides a pocket that perfectly matches and rewards the TS’s geometry and negative charge

thus lowers energy required to reach it