enzyme mechanisms 2006ppt

7/27/2019 Enzyme Mechanisms 2006ppt

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ENZYME MECHANISMS

Today we will consider aspects of the spectacular ability of enzymes to catalyze

chemical reactions. We will cover:

1) General Aspects. Enzymes catalyze reactions by lowering the activation

energy barrier for the reaction and often providing alternate pathways for a

reaction to occur. We will discuss four general categories of catalytic mechanisms

including a) transition state stabilization, b) general acid-base catalysis, c)

covalent catalysis and d) metal ion catalysis.

2) Classifications of Enzymes. Six different general classes of enzymes will be

discussed in order to provide a conceptual framework for enzymes that will be

covered in the course.

3) Proteases. As examples of enzymes with important medical consequences,we will discuss the properties and mechanisms of proteases, including four major

types of proteases. We will finish with a discussion of the mechanistic aspects of

catalysis by serine proteases.

Reading: Lippincott Chapter 5, sections I - IV


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I. General Aspects of enzymatic reactions and transition statesConsider a reaction A + B P + Q

where A + B react through transition state, X, to form products P + Q. K is theequilibrium constant between A + B and X and k' is the rate constant forconversion of X to P + Q.

A + B P + QK

k'

A + B

P + Q

G reaction

G

G

Reaction coordinate

The minimum energy pathway of the reactiois shown in the reaction coordinate, o r

transition state diagram, at left. Chemicalconversion ofA + B to P + Q proceedsthrough a transition state which is theleast stable (least probable, highest freeenergy) species along the pathway.Molecules that ac hieve the ac tivation energy,G, can go on to react while molecules thatfail to achieve the transition state f all back to

the ground state.

The transition state, X, is metastable with only a transient existence. The less stablethe transition state, the more difficult it is for a reaction to proceed.


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The smaller the difference in free energy of the reactants and the transition state, the faster thereaction proceeds. Enzymatic rate accelerations are achieved by lowering the activationbarrier between reactants and the transition state, thereby increasing the fraction ofreactants able to achieve the transition state. Enzymes catalyze reactions by eitherstabilizing the normal transition state or providing an alternative pathway from

reactants to products.

a) Transition state

stabil ization by induced fi t

b) Acid-base catalysis

c) Covalent Catalysis

d) Metal ion catalysis


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No catalysis is obtained by just binding substrate tightly!


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b) General acid-base

catalysis:Often used in the hydrolysis of

ester/ peptide bonds, phosphate

group reactions, addition to

carbonyl groups, etc.

An enzyme avoids unstable

charged intermediates inreaction (which would have high

free energies) by having groups

appropriately located to:

donate a proton (act as a

general acid), or

accept a proton (abstract a

proton, act as ageneral base)

H C

NH

COO

CH C

O

O-2

-

+3

Aspartic acid

Amino Acid pK a

3.90

-COOH

H C

NH

COO

CH C

O

O-2

-

+3

Glutamic acid 4.07

-COOH

CH2

H C

NH

COO

CH2

-

+3

Histidine 6.04

imidazole

N

N

H C

NH

COO

CH SH2

-

+3

Cysteine 8.33

sulfhydryl

H C

NH

COO

CH2

-

+3

Tyrosine 10.13

phenol

OH

H C

NH

COO

CH CH2

-

+3

Lysine 10.79

-amino

CH2 2

NH3+

What side-chains can act donate

or accept protons?


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Biologically important

nucleophilic groups:

Hydroxyl group R-OH R-O: + H+-

Sulfhydryl group R-SH

Adapted from Voet & Voet,Biochemistry

R-S:- + H+

R-NH3+ R-NH2 + H+Amino group

HN NH+

R

HN N:

R

+ H+Imidazole group

Nucleophilic form

R-NH2 + C=O

Biologically important

electrophiles:

C=OH+ Mn+

Protons Metal IonsCarbonyl carbon

c) Covalent catalysis (also sometimes called nucleophilic catalysis):(transient formation of a catalyst-substrate covalent bond)


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d) Metal ion catalysis:

Metal ions are often used for one or more of the following:* binding substrates in the proper orientation

* mediating oxidation-reduction reactions

* electrostatically stabilizing or shielding negative

charges (electrostatic catalysis)

Metalloenzymes contain tightly bound metal ions: (usually Fe+2,

Fe+3, Cu+2, Zn+2, or Mn+2)

Metal-activated enzymes contain loosely bound metal ions:(usually Na+, K+, Mg+2, or Ca+2)


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Which of the following mechanisms is not used by enzymes

for catalysis?

A. Acid base catalysis

B. Induced fit of intermediateC. Providing complementary electrostatics

D. Binding of metal ions

E. Destabilizing the transition state

Which of the following residue side-chains could not

contribute to acid-base catalysis?

A. Lysine

B. Glutamate

C. Histidine

D. Phenylalanine

E. Tyrosine


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II. Enzyme Classes

Enzymes have been grouped into 6 general classes by The Enzyme Commission. These

include 1) Oxidoreductases, 2) Transferases, 3) Hydrolases, 4) Lyases, 5) Isomerases

and 6) Ligases. Since such classifications can aid recognition of enzymes, we will brieflydiscuss each of these groups and provide examples.

1) Oxidoreductases: This is a very broad class of enzymes that catalyze the manyoxidation-reduction reactions found in biochemical pathways. Oxidoreductases catalyze

reactions in which at least one substrate gains electrons, becoming reduced, and another

loses electrons, becoming oxidized.

An important subset of oxido-

reductases are the dehydrogenases

that accept and donate electrons as

hydride ions (H:-) or hydrogenatoms often using cofactors such as

NAD+/NADH as an electron donor

or acceptor. An example is Lactate

Dehydrogenase (LDH) (right).R R

NAD+ NADH

LDH

(From Marks Basic Medical Biochemistry A clinical approach)


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2) Transferases: This class of enzymes catalyzethe transfer of a specific functional group between

molecules. Important subsets of transferases include

a)Kinases that transfer phosphate groups,

usually from ATP to another molecule (such as

hexokinase and glucokinase, that phosphorylate

glucose and protein kinases that phosphorylate

protein hydroyxl groups), b) Aminotransferases (see

right) that transfer amino groups that are important in

amino acid metabolism, c) Acyltransferases thattransfer fatty acyl groups and d)

Glycosyltransferases, which transfer carbohydrate

residues.Aminotransferase reaction in which

Pyridoxal phosphate (PLP) is used as a

cofactor. (From Marks Basic Medical

BiochemistryA clinical approach)

3) Hydrolases: Hydrolysis reactions refer to the cleavage of bonds by the addition of awater molecule. A very important class of hydrolases are the proteases involved in

cleaving peptide bonds, as we will be discussing shortly


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4) Lyases: This class refers to those enzymes involved in cleaving bonds by means

other than hydrolysis or oxidation. Examples include aldolases (such as fructose

diphosphate aldolase, which is involved in glycolysis) and thiolases (such as -

ketoacyl-CoA thiolase involved in the breakdown of fatty acids). Lyases alsoinclude enzymes involved in elimination of groups from two adjacent carbon atoms

to form double bonds.

In glycolysis, fructose 1,6-

bisphosphate aldolase cleaves a

carbon-carbon bond in fructose

1,6-bisphosphate. (From Marks

Basic Medical BiochemistryA

clinical approach)


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5) Isomerases: At many stages of metabolism,rearrangement of the atoms of a molecule is

required to create an isomer of the starting

compound. Enzymes generally catalyzing the

rearrangement of the bond structure are calledisomerases, while those specifically catalyzing

the movement of a phosphate from one group to

another are known as mutases. For example,

triose phosphate isomerase (right) catalyzes the

interconversion between dihydroxyacetone

phosphate and D-glyceraldehyde 3-phosphate,

which is essential for continuing glycolysis

following splitting of six carbon sugars into two

three carbon sugars by fructose diphosphate

aldolase

6) Ligases: Ligases are involved in synthesizing bonds between carbon atoms and

carbon, nitrogen, oxygen or sulfur atoms in reactions that are coupled to the cleavage of

the high energy phosphate of ATP or another nucleotide. Pyruvate carboxylase, a key

enzyme in gluconeogenesis, is one important ligase in metabolism.

(From Marks Basic Medical

BiochemistryA clinical approach)


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Proteases cleave peptide bonds by the addition of water. To

what class of enzymes do they belong?

A. Oxidoreductases

B. Ligases

C. Hydrolases

D. Lyases

E. Isomerases

15


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III. Proteases - Enzymes that specifically cut other proteins and

are important in regulation


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(usually His)

(Ser, Cys or Thr)

(Asp or Glu)

(metal)


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Protease classification

1) Cysteine Proteases

2) Aspartyl Proteases

3) Metalloproteases

4) Serine Proteases


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1) Cysteine Proteases

MECHANISM: In cysteine proteinases, catalysis proceeds

through the formation of a covalent intermediate and involves a

cysteine and a histidine residue. The essential Cys and His play the

same role as Ser and His respectively in Serine proteases as

discussed later. The nucleophile is a thiolate ion that is stabilized

through the formation of an ion pair with neighbouring

imidazolium group of His. The attacking nucleophile is the

thiolate-imidazolium ion pair in both steps.

EXAMPLES:

Medically interesting cysteine proteases include:

- mammalian enzymes such as cathepsins B and L, which are involved in cancer growth

and metastasis, and cathepsin K, which is important for bone degradation an osteoporosis.- Cruzipain and cruzain from Trypanosoma cruzi, which cause Chagas' disease, a

permanent infection that affects more than 25 million people annually in South America and

causes more than 45,000 deaths per year, and falcipain, fromPlasmodium falciparum,

which causes malaria.

- Caspases, which are key mediators of apoptosis.

2) A


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2) Aspartyl Proteases

MECHANISM: In contrast to cysteine (and serine) proteases,

catalysis by aspartic proteinases do not involve a covalent

intermediate, even though a tetrahedral intermediate istransiently formed. Rather, nucleophilic attack is achieved by

two simultaneous proton transfers: one from a water molecule to

one of the two carboxyl groups and a second one from the

carbonyl oxygen of the substrate with the concurrent CO-NH

bond cleavage. This general acid-base catalysis, which may be

called a "push-pull" mechanism leads to the formation of a noncovalent neutral tetrahedral intermediate

EXAMPLES:

Plasmepsin, which is produced in the parasite that causes malaria, is part of a closely related

group of enzymes known as aspartyl proteases. Plasmepsin is believed to play a key role in thedigestion of the human host's hemoglobin, the major nutrient source for the parasite.

HIV proteasepermits viral maturation.

BACE, an aspartyl protease involved in the amyloid peptide generation of Alzheimer's

disease.

3) M ll (Z )


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3) Metalloproteases (Zn)

MECHANISM: Many enzymes contain the sequence HEXXH,

which provides two histidine ligands for binding of zinc. A third Zn

ligand is either a glutamic acid (thermolysin, neprilysin, alanylaminopeptidase) or a histidine (astacin, serralysin). Other families

exhibit a distinct mode of binding of a Zn ion. The catalytic

mechanism involves formation of a non covalent tetrahedral

intermediate after the attack of a zinc-bound water molecule on the

carbonyl group of the scissile bond. This intermediate can be further

decomposed by transfer of the glutamic acid proton to the leavinggroup.

EXAMPLES:

Matrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the

degradation of extracellular matrix components such as collagen, laminin and proteoglycans.

These enzymes are involved in normal physiological processes such as embryogenesis and tissueremodeling and may play an important role in arthritis, periodontitis, and metastasis.

ACE is a metalloprotease that catalyses the conversion of angiotensin I into angiotensin II, which

leads to vasoconstriction. ACE inhibitors, were originally used as antihypertensives, but have

significantly improved the treatment of other cardiovascular diseases and are now used to treat

heart failure and even prevent heart attacks in at-risk patients.


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4) Serine Proteases

MECHANISM:The key active site groups are Ser, His and Asp. These groups are in the same orientation in all

the serine proteases. Their roles are basically as follows: the imidazole (His) acts as a general

base-general acid, first to activate theserine OH for nucleophilic catalysis, then the leaving

group (by general acid cat.), then as a general base it activates water to attack the covalent

acyl-enzyme intermediate. TheAsp serves to orient the His side chain and to provide an

appropriate electrostatic environment.

EXAMPLES:

Trypsin and Chymotrypsin are digestive enzymes in the small intestine

Subtilisin is a bacterial serine protease that is used in laundry detergents

TADG-14 is a novel extracellular serine protease that has been identified and cloned from

ovarian carcinoma. It is uniquely expressed in ovarian cancer, both in early stage and overtcarcinomas. It is seldom or not at all expressed in normal adult tissues and has not been

detected in other fetal tissues. It offers the potential as a target for therapeutic intervention

through down-regulation of its protease activity.

NS3/4A is a serine protease in Hepatitis C (HCV) that is important in viral maturation.

Factor VIIa, Factor Xa, and thrombin are serine proteases in blood coagulation pathway


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Trypsin and Chymotrypsin are very well studied serine proteases whose

structures and mechanisms are well understood

They catalyze the hydrolysis of internal peptide bonds (thus an endoprotease). Trypsin

cleaves on the carboxyl side of basic side chains (Lys, Arg), whereaschymotrypsin cleaves on the carboxyl side of aromatics (Phe, Tyr, Trp)

The active site consists of a catalytic triad:

1) Serine, to which the substrate binds

2) Histidine, which has the ability to donate and accept protons.

3) Aspartate, which has the ability to accept protons.

These residues are polar (hydrophilic) so would not ordinarily be

found on the "interior of a protein.

Though they are in close proximity in the 3D structure, they are not adjacent in the

primary sequence (Ser-195, His-57, Asp-102).

102


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First stage in peptide bond hydrolysis: acylation. Hydrolysis of the peptide bond starts with an

attack by the oxygen atom of the Ser195 hydroxyl group on the carbonyl carbon atom of the

susceptible bond. The carbon-oxygen bond of this carbonyl group becomes a single bond, and

the oxygen atom acquires a net negative charge. The four atoms now bonded to the carbonyl

carbon are arranged as a tetrahedron. Transfer of a proton from Ser195 to His57 is facilitated

by Asp102 which (i) precisely orients the imidazole ring of His57 and (i i) partly neutralizes the

positive charge that develops on His57 during the transition state. The proton held by the

protonated form of His57 is then donated to the nitrogen atom of the peptide bond that is

cleaved. At this stage, the amine component is hydrogen bonded to His57, and the acid

component of the substrate is esterified to Ser195. The amine component diffuses away.

Oxyanion

hole


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Second stage in peptide hydrolysis: deacylation. The acyl-enzyme intermediate

is hydrolyzed by water. Deacylation is essentially the reverse of acylation with

water playing the role as the attacking nucleophile, similar to Ser195 in the first

step. First, a proton is drawn away from water. The resulting OH- attacks the

carbonyl carbon of the acyl group that is attached to Ser195. As in acylation, atransient tetrahedral intermediate is formed. His57 then donates a proton to the

oxygen atom of Ser195, which then releases the acid component of the substrate,

completing the reaction.

Oxyanion

hole


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(From Marks Basic Medical Biochemistry A clinical approach)


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Activation Strategies for three classes of Proteases

From: Berg, Tymoczko and Stryer: Biochemistry, 5th edition


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How is specificity obtained among trypsin-like serine proteases?


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Are all serine proteases evolutionarily related? (NO!)

Catalytic triad: Ser 195, His 57, Asp 102 Catalytic triad: Ser 221, His 64, Asp 32


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Which of the following peptides would trypsin cleave?

A. Asp-Leu-Trp-Ala-Leu-Met-Tyr

B. Asn-Thr-Ser-Asp-Ala-Leu-Gly

C. His-Ala-Asp-Val-Asn-Gln-Ala

D. Val-Ala-Val-Lys-Ser-Gly-Phe

E. Phe-Ala-Trp-Ser-Ile-Ala-Gly


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The physiological importance of many proteases has made

them an extremely active objects of current research:

Highly selective small organic compounds are being

investigated as therapeutic leads against many proteases.

This is one of the hottest areas of drug development in thebiotech and pharmaceutical industry

Many compounds are in preclinical or clinical trials.

Many of these compounds will likely be drugs by the time

you are doctors!


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Summary Points on Enzyme Catalys is :

1) Understand what is meant by transition state stabilization.

2) Know which amino acid residues can be involved in acid-base catalysis.

3) Understand that covalent catalysis results from the transient formation of a covalent bond

involving a nucleophile and an electrophile.

4) Recognize the general classes of enzymes, including oxidoreductases, transferases,

hydrolases, lyases, isomerases and ligases.

5) Recognize the physiological importance of Proteases.

6) Understand that proteases use nucleophilic attack by either covalent catalysis or

activation of a water molecule, along with acid-base catalysis.

7) Know what is meant by a serine protease.

8) Have a general understanding of covalent catalysis by Trypsin.

9) Understand that the specificity pocket allows different members of the trypsin family of

proteases to use the same catalytic mechanism for proteolysis but with very different

sequence specificities.

enzyme mechanisms 2006ppt

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