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Enzymes. Most biological catalysts are ____________ (some REALLY COOL ONES are folded RNAs!!!) Catalysts - Catalyst does not alter equilibrium. Nonenzymatic reaction rate (s -1 ). Enzymatic reaction rate (s -1 ). Enzyme. Rate enhancement. Carbonic anhydrase. 1.3 x 10 -1.

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slide1

Enzymes

Most biological catalysts are ____________

(some REALLY COOL ONES are folded RNAs!!!)

Catalysts -

Catalyst does not alter equilibrium

Nonenzymatic

reaction rate (s-1)

Enzymatic

reaction rate (s-1)

Enzyme

Rate enhancement

Carbonic

anhydrase

1.3 x 10-1

1 x 106

7.7 x 106

Triose phosphate

isomerase

4.3 x 10-6

4300

1 x 109

Staphlococcal

nuclease

1.7 x 10-13

95

5.6 x 1014

slide2

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

slide3

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 (Cu2+, Fe3+, Zn2+)

trace amounts of metal needed in our diets

slide4

Enzymes

Cofactors

COFACTORS can be organic or metalloorganic molecules --> COENZYMES

Examples:

NAD+

Heme

Holoenzyme =

Apoenzyme (inactive) + cofactor/coenzyme/metal ions

slide5

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

Human

Disease

Coenzyme

Reaction mediated

Vitamin source

Cobalamin

coenzymes

Alkylation

Cobalamin (B12)

Pernicious anemia

Flavin

coenzymes

Oxidation-reduction

Riboflavin (B2)

rare

Nicotinamide

coenzymes

Oxidation-reduction

Nicotinamide (niacin)

Pellagra

Pyridoxal

phosphate

Amino group transfer

Pyridoxine (B6)

rare

Tetrahydrofolate

One-carbon group transfers

Folic acid

Megaloblastic anemia

Thiamine

pyrophosphate

Aldehyde transfer

Thiamine (B1)

Beriberi

slide6

Enzymes

Substrate specificity

Types of complementarity between enzyme and substrate:

Substrate binding sites undergo conformational change when substrate binds

slide7

Enzymes

Enzyme undergoes conformational change when substrate binds - induced fit

Substrate

a

c

b

a

+

c

b

ES complex

a

c

b

Enzyme

slide8

Enzyme-substrate complementarity

Dihydrofolate reductase-NADP+(red)-tetrahydrofolate (yellow)

slide9

Enzymes

Stereospecific

Why?

Inherently chiral (proteins only consist of L-amino acids) so form asymmetric active sites

Example: Protein enzyme Yeast Alcohol dehydrogenase (YADH)

O

YADH

CH3CH2OH + NAD+

CH3CH + NADH + H+

Ethanol

Acetaldehyde

slide10

Enzymes

Stereospecific

Yeast Alcohol dehydrogenase (YADH) is stereospecific

1. If YADH reaction uses deuterated ethanol, NAD+ is deuterated to form NADD

O

D

H

NADD

O

NAD+

C

H

YADH

NH2

C

NH2

N

+

N

O

R

R

+ CH3CD + H+

+ CH3CD2OH (ethanol)

(acetaldehyde)

2. Isolate NADD and use in reverse reaction to reduce normal acetaldehyde, deuterium transferred from NADD to acetaldehyde to form ethanol

O

OH

D

H

YADH

C

Hpro-S

Dpro-R

C

NH2

N

CH3

O

R

+NAD+

+ CH3CH + H+

3. Enantiomer of ethanol - none of deuterium is transferred from this isomer of ethanol to NAD+ in the reverse reaction

OH

Dpro-S

Hpro-R

C

CH3

slide11

Enzyme activity

Dependent on:

[metal ion], pH, temperature, [enzyme], [substrate]

slide12

Enzymes

E + S ES E + P

G’˚ < 0; favorable

slide13

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

slide14

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

R

R

+

-

-

C

O

H A

H

A

C

O

C

O

H

CH2

-

CH2

CH2

H

H

A-

H

+

General base catalysis - partial proton abstraction by a base lowers free energy of reaction’s transition state

Keto

Transition state

Enol

R

R

-

R

C

O

C

O

C

O

H

-

CH2

CH2

CH2

H

H+

H

+

H B

B

+

B

slide15

Enzymes

General acid-base catalysis

slide16

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

slide17

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

SA-SB + N: SA -N + SB SA + N: + SB

H2O

Example: Decarboxylation of acetoacetate (catalyst contains primary amine)

acetone

acetoacetate

O

O

O

CH3

C

CH2

C

CH3

C

CH3

O-

RNH2

+ RNH2

+ OH-

OH-

CO2

R

H

+

R

H

R

H

..

+

+ H+

N

O

N

N

CH3

C

CH2

C

CH3

C

CH2

CH3

C

CH3

O-

SCHIFF BASE

(IMINE)

slide18

Enzymes

Covalent Catalysis

R

+

HN

NH

Some amino acids with nucleophilic groups

ROH Serine

RSH Cysteine

RNH3+ Lysine

Histidine

slide19

Enzymes

Metal Ion Catalysis

One-third of all known enzymes require metal ions --> metalloenzymes

Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, Co2+ (sometimes Na+, K+, Mg2+, Ca2+)

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

slide20

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 = Ser195, Asp102, His57

slide22

Enzymes:

Chymotrypsin

general base

general acid

general acid

Covalent intermediate

general base

slide23

Enzymes:

Chymotrypsin

slide24

Enzymes:

Chymotrypsin

slide25

Enzymes:

Chymotrypsin

slide26

Enzymes:

Chymotrypsin

slide27

Enzymes:

Chymotrypsin and other serine proteases

slide28

Enzymes:

Enolase

catalyzes reaction step of glycolysis

reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate

Metal ion catalysis, general acid-base, transition state stabilization

Lys345 = general base, abstracts proton from C-2 of 2-phosphoglycerate

Glu211 = general acid, donates proton to -OH leaving group

slide29

Enzymes:

Enolase

Metal ion catalysis

2 Mg2+ ions interact with 2-phosphoglycerate making the C-2 proton more acidic (lower pKa) and easier to abstract