The Organic Chemistry of Enzyme-Catalyzed Reactions

Revised Edition

Professor Richard B. SilvermanDepartment of Chemistry

Department of Biochemistry, Molecular Biology, and Cell BiologyNorthwestern University

The Organic Chemistry of Enzyme-Catalyzed Reactions

Chapter 1

Enzymes as Catalysts

For published data regarding any enzyme see:http://www.brenda-enzymes.info/Nomenclature Enzyme Names EC Number Common/ Recommended Name Systematic Name Synonyms CAS Registry Number

Reaction & Specificity Pathway Catalysed Reaction Reaction Type Natural Substrates and Products Substrates and Products Substrates Natural Substrate Products Natural Product Inhibitors Cofactors Metals/ Ions Activating Compounds Ligands

Functional Parameters Km Value Ki Value pI Value Turnover Number Specific Activity pH Optimum pH Range Temperature Optimum Temperature Range

Isolation & Preparation Purification Cloned Renatured Crystallization

Organism- related information Organism Source Tissue Localization

Stability pH Stability Temperature Stability General Stability Organic Solvent Stability Oxidation Stability Storage Stability

Enzyme Structure Sequence/ SwissProt link 3D-Structure/ PDB link Molecular Weight Subunits Posttranslational Modification

Disease & References Disease References

Application & Engineering Engineering Application

What are enzymes, and how do they work?

• First “isolation” of an enzyme in 1833• Ethanol added to aqueous extract of malt • Yielded heat-labile precipitate that was

utilized to hydrolyze starch to soluble sugar; precipitate now known as amylase

• 1878 - Kühne coined term enzyme - means “in yeast”

• 1898 - Duclaux proposed all enzymes should have suffix “ase”

• Enzymes - natural proteins that catalyze chemical reactions

• First enzyme recognized as protein was jack bean urease

• Crystallized in 1926• Took 70 more years (1995), though, to obtain

its crystal structure

• Enzymes have molecular weights of several thousand to several million, yet catalyze transformations on molecules as small as carbon dioxide and nitrogen

• Function by lowering transition-state energies and energetic intermediates and by raising the ground-state energy

• Many different hypotheses proposed for how enzymes catalyze reactions

• Common link of hypotheses: enzyme-catalyzed reaction always initiated by the formation of an enzyme-substrate (or ES) complex in a small cavity called the active site

• 1894 - Lock-and-key hypothesis - Fischer proposed enzyme is the lock into which the substrate (the key) fits

• Does not rationalize certain observed phenomena:

Compounds having less bulky substituents often fail to be substrates

Some compounds with more bulky substituents bind more tightly

Some enzymes that catalyze reactions between two substrates do not bind one substrate until the other one is bound

1958 - Induced-fit hypothesis proposed by Koshland:

When a substrate begins to bind to an enzyme, interactions induce a conformational change in the enzyme

Results in a change of the enzyme from a low catalytic form to a high catalytic form

Induced-fit hypothesis requires a flexible active site

Concept of flexible active site stated earlier by Pauling (1946):

Hypothesized that an enzyme is a flexible template that is most complementary to substrates at the transition state rather than at the ground state

Therefore, the substrate does not bind most effectively in the ES complex

As reaction proceeds, enzyme conforms better to the transition-state structure

Transition-state stabilization results in rate enhancement

• Only a dozen or so amino acid residues may make up the active site

• Only two or three may be involved directly in substrate binding and/or catalysis

Why is it necessary for enzymes to be so large?• Most effective binding of substrate results

from close packing of atoms within protein• Remainder of enzyme outside active site is

required to maintain integrity of the active site• May serve to channel the substrate into the

active siteActive site aligns the orbitals of substrates and

catalytic groups on the enzyme optimally for conversion to the transition-state structure-- called orbital steering

• Enzyme catalysis characterized by two features: specificity and rate acceleration

• Active site contains amino acid residues and cofactors that are responsible for the above features

• Cofactor, also called a coenzyme, is an organic molecule or metal ion that is essential for the catalytic action

Specificity of Enzyme-Catalyzed Reactions• Two types of specificity: (1) Specificity of binding

and (2) specificity of reactionSpecificity of Binding

• Enzyme catalysis is initiated by interaction between enzyme and substrate (ES complex)

• k1, also referred to as kon, is rate constant for formation of the ES complex

• k-1, also referred to as koff, is rate constant for breakdown of the complex

• Stability of ES complex is related to affinity of the substrate for the enzyme as measured by Ks, dissociation constant for the ES complex

Ks =

E + S E . E . E + Pk2

k-1

k1

k-1

k1

S P

Scheme 1.1

kon

koff

Michaelis complex

When k2 << k-1,k2 called kcat (turnover number)Ks called Km (Michaelis-Menten constant)

Generalized enzyme-catalyzed reaction

kcat represents the maximum number of substrate molecules converted to product molecules per active site per unit of time; called turnover number

Table 1.1. Examples of Turnover Numbersa

Enzyme Turnover numberkcat (s-1)

papain 10carboxypeptidase 102

acetylcholinesterase 103

kinases 103

dehydrogenases 103

aminotransferases 103

carbonic anhydrase 106

superoxide dismutase 106

catalase 107

aEigen, M.; Hammes, G.G. Adv. Enzymol. 1963, 25, 1.

• Km is the concentration of substrate that produces half the maximum rate

• Km is a dissociation constant, so the smaller the Km the stronger the interaction between E and S

• kcat/Km is the specificity constant - used to rank an enzyme according to how good it is with different substrates

Upper limit for is rate of diffusion (109 M-1s-1)Km

kcat

How does an enzyme release product so efficiently given that the enzyme binds the transition state structure about 1012 times more tightly than it binds the substrate or products?

After bond breaking (or making) at transition state, interactions that match in the transition-state stabilizing complex are no longer present.

Therefore products are poorly bound, resulting in expulsion.

As bonds are broken/made, changes in electronic distribution can occur, generating a repulsive interaction, leading to expulsion of products

E • S complexFigure 1.1Non-covalent interactions

electrostatic(ionic)

C

O

O

+

RNH3

ion -dipo le R

C NH3

R'

Oδ

δ+

δipole-δipole R

C O

R'

Oδδδ

δ

H

H-bonδing

O

RC O HO

H

chargetransfer

A

D

D

A

hyδrophobic

ORC

O

∆Gº = -RTlnKeq

If Keq = 0.01, ∆Gº of -5.5 kcal/mol needed to shift Keq to 100

Specific Forces Involved in E•S Complex Formation

Figure 1.2

NH3 O

OH

CH3

COCH2

CH2

NMe3

+

+

δ−

δ+

δ−

δipole-δipoleδ+

ion-δipole

O

O

ionic

Examples of ionic, ion-dipole, and dipole-dipole interactions. The wavy line represents the

enzyme active site

H-bonds

A type of dipole-dipole interaction between X-H and Y: (N, O)

Figure 1.3

H-bonds

Hydrogen bonding in the secondary structure of proteins: a-helix and b-sheet.

Charge Transfer Complexes

• When a molecule (or group) that is a good electron donor comes into contact with a molecule (or group) that is a good electron acceptor, donor may transfer some of its charge to the acceptor

Hydrophobic Interactions

• When two nonpolar groups, each surrounded by water molecules, approach each other, the water molecules become disordered in an attempt to associate with the water molecules of the approaching group

• Increases entropy, resulting in decrease in the free energy (DG = DH-TDS)

van der Waals Forces

• Atoms have a temporary nonsymmetrical distribution of electron density resulting in generation of a temporary dipole

• Temporary dipoles of one molecule induce opposite dipoles in the approaching molecule

Binding Specificity

• Can be absolute or can be very broad• Specificity of racemates may involve E•S complex

formation with only one enantiomer or E•S complex formation with both enantiomers, but only one is converted to product

• Enzymes accomplish this because they are chiral molecules (mammalian enzymes consist of only L-amino acids)

Binding specificity of enantiomers

Scheme 1.2

EnzL + (R,S) EnzL + EnzLR Sdiastereomers

Resolution of a racemic mixture

• Binding energy for E•S complex formation with one enantiomer may be much higher than that with the other enantiomer

• Both E•S complexes may form, but only one E•S complex may lead to product formation

• Enantiomer that does not turn over is said to undergo nonproductive binding

Steric hindrance to binding of enantiomers

Figure 1.4

OOCNH

3

H

OOC NH3

H

A B

S R

Leu

Basis for enantioselectivity in enzymes

Reaction Specificity

Unlike reactions in solution, enzymes can show specificity for chemically identical protons

Figure 1.5

R R'

R R'

Ha

Hb

B

-

enzyme

Enzyme specificity for chemically identical protons. R and R on the enzyme are

groups that interact specifically with R and R, respectively, on the substrate.

Rate Acceleration

• An enzyme has numerous opportunities to invoke catalysis:– Stabilization of the transition state– Destabilization of the E•S complex– Destabilization of intermediates

• Because of these opportunities, multiple steps may be involved

Figure 1.6 1010-1014 fold typically

Catalyzed

Uncatalyzed

Reaction Coordinate

Free Energy (∆G)

A

Uncatalyzed

Enzyme Catalyzed

Reaction Coordinate

Free Energy (∆G)

B

E+S

E+P

ESEP

Effect of (A) a chemical catalyst and (B) an enzyme on activation energy

Enzyme catalysis does not alter the equilibrium of a reversible reaction; it accelerates attainment of the equilibrium

Table 1.2. Examples of Enzymatic Rate Acceleration

Enzyme Nonenzymatic rate knon (s-1)

Enzymatic rate kcat (s-1)

Rate acceleration kcat/knon

cyclophilina 2.8 x 10-2 1.3 x 104 4.6 x 105

carbonic anhydrasea 1.3 x 10-1 106 7.7 x 106

chorismate mutasea 2.6 x 10-5 50 1.9 x 106

chymotrypsinb 4 x 10-9 4 x 10-2 107

triosephosphateisomeraseb

6 x 10-7 2 x 103 3 x 109

fumaraseb 2 x 10-8 2 x 103 1011

ketosteroid isomerasea 1.7 x 10-7 6.6 x 104 3.9 x 1011

carboxypeptidase Aa 3 x 10-9 578 1.9 x 1011

adenosine deaminasea 1.8 x 1010 370 2.1 x 1012

ureaseb 3 x 10-10 3 x 104 1014

alkaline phosphataseb 10-15 102 1017

orotidine 5'-phosphatedecarboxylasea

2.8 x 10-16 39 1.4 x 1017

a Taken from Radzicka, A.; Wolfenden, R. Science 1995, 267, 90.b Taken from Horton, H.R.; Moran, L.A.; Ochs, R.S.; Rawn, J.D.; Scrimgeour,K.G. Principles of Biochemistry; Neil Patterson: Englewood Cliffs, NJ, 1993.

Mechanisms of Enzyme Catalysis

Approximation

• Rate enhancement by proximity• Enzyme serves as a template to bind the

substrates• Reaction of enzyme-bound substrates

becomes first order• Equivalent to increasing the concentration of

the reacting groups• Exemplified with nonenzymatic model studies

Scheme 1.3

CH3COAr

O O

CO

C

O

H3C+ CH3COO-

CH3+ ArO-

Second-order reaction of acetate with aryl acetate

OAr

O

O

O

-

OAr

O

O

O

-

OAr

O

O

O

-

OAr

O

O

O

O

O

-

Relative rate ( krel

)

1 M

-1

s

-1

220 s

-1

5.1 x 10

4

s

-1

2.3 x 10

6

s

-1

1.2 x 10

7

s

-1

Decreasing rotational and

translational entropy

+ CH3

COO

-

OAr

Effective Molarity (EM)

5.1 x 10

4

M

2.3 x 10

6

M

1.2 x 10

7

M

220 M

Table 1.3. Effect of Approximation on Reaction Rates

Covalent Catalysis

Scheme 1.4anchimeric assistance

Most commonCys (SH)Ser (OH)His (imidazole)Lys (NH2)Asp/Glu (COO-)

R Y

O

X X

R Y

O-OR

X

X ZR

O

Activated carbonyl

Z-

+

1.1

-Y-

Nucleophilic catalysis

X-

Scheme 1.5

SCl

SOH

S+

1.2

HO--Cl-

Anchimeric assistance by a neighboring group

Model Reaction for Covalent Catalysis

Scheme 1.6

Early evidence to support covalent catalysis

O

O

18O

O 18OCH3C

18O

18OH

O

OH

18O18

+Ar

H2O

H2O O-

(-ArO-)

General Acid/Base Catalysis

This is important for any reaction in which proton transfer occurs

Figure 1.7catalytic triad

The catalytic triad of a-chymotrypsin. The distances are as follows: d1 = 2.82 Å; d2 =

2.61 Å; d3 = 2.76 Å.

Scheme 1.7

HN

NH

NHR'

Ser OH

R1

O R2

O

R

N N H

His

-OOC Asp

Charge relay system for activation of an active-site serine residue in a-chymotrypsin

• pKa values of amino acid side-chain groups within the active site of enzymes can be quite different from those in solution

• Partly result of low polarity inside of proteinsMolecular dynamics simulations show interiors of these proteins have dielectric constants of about 2-3 (dielectric constant

for benzene or dioxane)• If a carboxylic acid is in a nonpolar region, pKa will

rise• Glutamate-35 in the lysozyme-glycolchitin complex

has a pKa of 8.2; pKa in solution is 4.5• If the carboxylate ion forms salt bridge, it is

stabilized and has a lower pKa

• Basic group in a nonpolar environment has a lower pKa

• pKa of a base will fall if adjacent to other bases

• Active-site lysine in acetoacetate decarboxylase has a pKa of 5.9 (pKa in solution is 10.5)

Two kinds of acid/base catalysis:

• Specific acid or specific base catalysis - catalysis by a hydronium (H3O+) or hydroxide (HO-) ion, and is determined only by the pH

• General acid/base catalysis - reaction rate increases with increasing buffer concentration at a constant pH and ionic strength

Figure 1.8Specific acid/base catalysis General acid/base catalysis

k k

[Buffer] [Buffer]

pH 7.9

pH 7.3

pH 7.9

pH 7.3

A B

Effect of the buffer concentration on (A) specific acid/base catalysis and (B)

general acid/base catalysis

Scheme 1.8

Specific Acid-Base Catalysis

O

C OEt

poornucleophileweak

electrophile

++H3C EtOHCH3COOHH2O

Hydrolysis of ethyl acetate

Scheme 1.9

Alkaline hydrolysis of ethyl acetate

O

COHH3C

O

COC2H5H3C

O

CO-H3C

+ +

strongnucleophile

C2H5O-

HO-

C2H5OH

Scheme 1.10

OC

OHH3C

OHC

OC2H5H3C

OHC

OC2H5H3C

OC

OC2H5H3C +

+

++

strongelectrophile

H3O+ H2OC2H5OH

Acid hydrolysis of ethyl acetate

Scheme 1.11

B+

H

R Y

O

H OHB:

Simultaneous acid and base enzyme catalysis

base catalysis

acid catalysis

Enzymes can utilize acid and base catalysis simultaneously

Simultaneous acid/base catalysis is the reason for how enzymes are capable of deprotonating weak carbon acids

Scheme 1.12

Simultaneous acid and base enzyme catalysis in the enolization of mandelic acid

Ph

HaHO OHb

OPh

O-

O

HaHO

Ph

HaHO OHb

OHc

Ph

HaHO OHb

OHcPh

HO

O-

OHb

Ph

HO

OHc

OHb

pKE = 18.6

+

+

pKa ~ 7.4pKa = 6.6

± Hc+ ± Ha

+

pKE = 15.4

pKa = 22.0

± Ha+

pKa ~ -8

± Hc+

pKa = 3.4

± Hb+

1.3 1.4

1.51.6

• Low-barrier hydrogen bonds - short (< 2.5Å), very strong hydrogen bonds

• Stabilization of the enolic intermediate occurs via low-barrier hydrogen bonds

X R

H

O

H

B:

BH

X R

HO

:B

B+H

R

O

R = H, alkyl, SR'

O–

OX

H HM+

BH

O–

OXH

M+

B

O

O

H

M+

BH

B

H

BB:

B:BH

-HX

A

B

Scheme 1.13

low-barrier H-bond

“weak” base

“strong” acid

“strong” base

“weak” acid

low-barrier H-bond

stronger acid needed

One-base mechanism syn-elimination

carboxylic acids

Simultaneous acid and base enzyme catalysis in the 1,4-elimination of b-substituted (A) aldehydes, ketones, thioesters and (B) carboxylic acids

Two-base mechanism anti-elimination

Scheme 1.14

ElcB mechanism - not relevant

X R

H

O

H

X R

O

B+H

R

O

B:

Base catalyzed 1,4-elimination of b-substituted carbonyl compounds via an enolate

intermediate (ElcB mechanism)Needs acid or metal catalysis

Alternative to Low-Barrier Hydrogen Bond

Scheme 1.15

R

H

O

H

R' R

O

R'

H

B: B+H

Electrostatic enzyme catalysis in enolization

Electrostatic Catalysis

Scheme 1.16

oxyanion hole

HN

NH

HN

NH

NH

HN

NH

HN

O

O

O O

OO

O

R"

R'

RR"

R'

RO

++

also could be aH bond or dipole

Electrostatic stabilization of the transition state

Desolvation

• Exposes substrate to lower dielectric constant environment

• Exposes water-bonded charged groups for electrostatic catalysis

• Destabilizes the ground state

The removal of water molecules at the active site on substrate binding

Scheme 1.17

Strain Energy

k1.8

k1.7 = 108

OP

O HO

-OP

O

O- OO-

-OO

PO

O--O

CH3CH3

OP

O-O

-O

CH3

CH3

HO

1.7 1.8

-OH -OH

Alkaline hydrolysis of phosphodiesters

Figure 1.9

Induced Fit Hypothesisputting strain energy into the substrate

Figure 1.10

Energetic Effect of Enzyme Catalysis

Importance of ground state destabilization

H

Lys252

NH2

NH2

O

COO-

NH2

O

COO-

NH

NH-Lys252

COO-

NH2

COO-B:

NH

NH-Lys252

COO-

NH2

COO-

NH

COO- COO-

H

NH2

NH-Lys252

B

B:H

:B

NH

COO- COO-

NH2

HNH

COO- COO-

NH2

+ +

..

+

+

+

..

:

ZnB(Cys)4

Lys252

NH

NH2

O

COO-

ZnB(Cys)4

H :B

Lys252

NH

NH2OH

COO-

ZnB(Cys)4

Lys252

NH

H2N

COO-

NH2

O

COO- Lys252

NH

N

COO-

HH

:B (X)3ZnA

HO

(X)3ZnA

HO

H

(X)3ZnA

HO

(X)3ZnA

HO

(X)3ZnA

HO

strain energyelectrostatic catalysis

approximationcovalent catalysis

base catalysis

strain energyelectrostatic catalysis

base catalysis

base catalysis

acid catalysis

base catalysis

base catalysis

approximation

approximation

(X3)ZnA (X3)ZnA

Mechanisms of Enzyme Catalysis - porphobilinogen synthase