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Enzymes

Most biological catalysts are ____________ (some REALLY COOL ONES are folded RNAs!!!) Catalysts Catalyst does not alter equilibrium Enzyme Carbonic anhydrase Nonenzymatic reaction rate (s 1.3 x 10 -1 Triose phosphate isomerase 4.3 x 10 -6 -1 ) Enzymatic reaction rate (s -1 ) 1 x 10 6 4300 Rate enhancement 7.7 x 10 6 1 x 10 9 Staphlococcal nuclease 1.7 x 10 -13 95 5.6 x 10 14

Enzymes

E + S ES E + P Highly specific Reaction occurs in ___________ of enzyme Substance acted upon = __________ Resulting species = _____________ Enzyme acts on forward and reverse reactions Activity depends on protein’s native structure Regulated - by concentrations of substrate and substances other than substrate

Enzymes

Cofactors

Functional groups of protein enzymes are involved in acid-base reactions, covalent bond formation, charge-charge interactions BUT they are less suitable for oxid-reduc and group-transfer reactions SO they use

__________________

(inorganic ions)

COFACTORS

may be metal ions (Cu 2+ , Fe 3+ , Zn 2+ ) trace amounts of metal needed in our diets

Enzymes

Cofactors

COFACTORS can be organic or metalloorganic molecules --> COENZYMES Examples: NAD + Heme Holoenzyme = Apoenzyme (inactive) + cofactor/coenzyme/metal ions

Enzymes

Coenzymes

Coenzymes must be regenerated Many vitamins are coenzyme precursors Vitamins must be present in our diets because we cannot synthesize certain parts of coenzymes Coenzyme Reaction mediated Vitamin source Human Disease Cobalamin coenzymes Alkylation Cobalamin (B12) Pernicious anemia Flavin coenzymes Oxidation-reduction Riboflavin (B2) rare Nicotinamide coenzymes Oxidation-reduction Pyridoxal phosphate Amino group transfer Tetrahydrofolate One-carbon group transfers Thiamine pyrophosphate Aldehyde transfer Nicotinamide (niacin) Pyridoxine (B6) Folic acid Thiamine (B1) Pellagra rare Megaloblastic anemia Beriberi

Substrate specificity

Enzymes

Types of complementarity between enzyme and substrate: Substrate binding sites undergo conformational change when substrate binds

Enzymes

Enzyme undergoes conformational change when substrate binds -

induced fit Substrate a b c + a b Enzyme c a b ES complex c

Enzyme-substrate complementarity Dihydrofolate reductase-NADP + (red)-tetrahydrofolate (yellow)

Enzymes

Stereospecific

Why? Inherently chiral (proteins only consist of L-amino acids) so form asymmetric active sites Example: Protein enzyme Yeast Alcohol dehydrogenase (YADH) YADH O CH 3 CH 2 OH + NAD + Ethanol CH 3 CH + NADH + H Acetaldehyde +

Enzymes

Stereospecific

Yeast Alcohol dehydrogenase (YADH) is stereospecific 1. If YADH reaction uses deuterated ethanol, NAD + NADD H O C

NAD +

YADH D + N NH 2 is deuterated to form N R H O C NH O 2

NAD D

R + CH 3 C D 2 OH (ethanol) + CH 3 C D + H (acetaldehyde) + 2. Isolate NAD D and use in reverse reaction to reduce normal acetaldehyde, deuterium transferred from NAD D to acetaldehyde to form ethanol O D H YADH OH C NH 2 H

pro-S

C D

pro-R

N R O CH 3 +NAD + + CH 3 CH + H + 3. Enantiomer of ethanol - none of deuterium is transferred from this isomer of ethanol to NAD + in the reverse reaction OH D

pro-S

C H

pro-R

CH 3

Enzyme activity Dependent on: [metal ion], pH, temperature, [enzyme], [substrate]

E + S ES

Enzymes

E + P  G’˚ < 0; favorable

Enzymes

Enzymes affect reaction rates, not ____________ Catalysts enhance reaction rates by lowering __________________ Rate is set by activation energy  G ‡ Higher activation energy --> _____________ Overall rate of reaction is determined by step with highest activation energy --> rate-limiting step

Enzymes

General acid-base catalysis

General acid catalysis - partial proton transfer from an acid lowers free energy of reaction’s transition state Keto Transition state Enol R C CH 2 H O

H A

R C CH 2   O H + +

-

H R C CH 2 O A H General base catalysis - partial proton abstraction by a base lowers free energy of reaction’s transition state Keto Transition state Enol R C CH 2 H

O R C CH 2   O H +

+ H+ R C CH 2 O H B H

Enzymes

General acid-base catalysis

Enzymes

General acid-base catalysis

Example: Ribonuclease A (RNase A) digestive enzyme secreted by pancreas into small intestine hydrolyzes RNA rate depends on pH, suggesting involvement of ionizable residues His12 and His119

Enzymes

Covalent Catalysis

Transient covalent bond formed between E and S Accelerates reaction rate through transient formation of a catalyst-substrate covalent bond Usually covalent bond is formed by the reaction of a nucleophilic group on the catalyst with an electrophilic group on the substrate --> nucleophilic catalysis S A -S B + N: S A N + S B S A + N: + S B H 2 O Example: Decarboxylation of acetoacetate (catalyst contains primary amine) O acetoacetate O acetone O CH 3 C CH 2 C O CH 3 C CH 3

+ RNH 2 RNH 2 OH + OH R + N H

O CH 3 C CH 2 C O-

SCHIFF BASE (IMINE)

CO 2

R ..

N H

+ H + CH 3 C CH 2

R + N H

CH 3 C CH 3

Enzymes

Covalent Catalysis

Some amino acids with nucleophilic groups

ROH RSH RNH

+

Serine Cysteine Lysine Histidine

Enzymes

Metal Ion Catalysis

One-third of all known enzymes require metal ions --> metalloenzymes Fe 2+ , Fe 3+ , Cu 2+ , Zn 2+ , Mn 2+ , Co 2+ (sometimes Na + , K + , Mg 2+ , Ca 2+ ) Metal bound to enzyme (or substrate) What can it do?

help orient substrate (or enzyme) for reaction stabilize charged reaction transition state mediate oxidation-reduction reactions (change metal’s oxidation state) Voet, p. 295 11-11, scheme

Enzymes: Chymotrypsin

Serine protease, very reactive serine residue in enzyme Digestive enzyme synthesized by pancreas Catalyzes cleavage of peptide bonds adjacent to aromatic amino acids Transition state stabilization General acid-base catalysis and covalent catalysis Catalytic triad = Ser 195 , Asp 102 , His 57

Enzymes: Chymotrypsin

general base general acid Covalent intermediate general base general acid

Enzymes: Chymotrypsin

Enzymes: Chymotrypsin and other serine proteases

Enzymes: Enolase

catalyzes reaction step of glycolysis reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate Metal ion catalysis, general acid-base, transition state stabilization Lys 345 Glu 211 = general base, abstracts proton from C-2 of 2-phosphoglycerate = general acid, donates proton to -OH leaving group

Enzymes: Enolase

Metal ion catalysis 2 Mg 2+ ions interact with 2-phosphoglycerate making the C-2 proton more acidic (lower p

K a

) and easier to abstract