Enzyme Specificity

Lecture 3

Objective

To understand Specificity of enzymes

Specificity means:

• Ability of an enzyme to catalyse a specific reaction and NO others

The active site

• The active site, is a special region where catalysis occurs

• Occupies small part of enzyme volume

• The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

Closer Look at Active site

•AS is not a point or line on enzyme

•It is a region in enzyme molecule where catalysis occurs

•AS has binding site and a catalytic site

•AS has a 3D Structure; residues widely separated in the primary structure are brought closer in the AS

•Clefts or crevices

AS: General Characteristics

• Substrates bound by multiple weak interactions• AS include both polar and nonpolar amino acids and

create hydrophilic and hydrophobic microenvironment

• Specificity depends on precise arrangement of atoms in active site

• Substrates bound by multiple weak interactions• Specificity depends on precise arrangement of

atoms in active site

Enzyme Amino acid in active site

Hexokinase His

Phosphoglucomutase Ser

Trypsin Ser, His

Carbonic anhydrase CysteineCarboxypeptidase His, Arg, Tyr

Thrombin Ser, His

Aldolase Lys

Chymotrypsin Ser, his

Choline esterase Ser

Cofactors

• An additional non-protein molecule that is needed by some enzymes to help the reaction

• Tightly bound cofactors are called prosthetic groups

• Cofactors that are bound and released easily are called coenzymes

• Many vitamins are coenzymes

Nitrogenase enzyme with Fe, Mo and ADP cofactorsH.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES

STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS

IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

The Substrate

• The substrate of an enzyme are the reactants that are activated by the enzyme

• Enzymes are specific to their substrates• The specificity is determined by the active site

• Enzyme specificity can be for the type of reaction it catalyses or for its choice of substrates

• Substrate has a bond or linkage that can be attacked by the enzyme

Active site

At the active site, functional groups of the enzyme interact with substrate (eg: RNase A)

This interaction may weaken one of its bonds or participate in a reaction by transforming an electron or proton

(eg: catalytic mechanism of Serine proteases)

• Enzyme and substrate binding releases ‘binding energy’.

• This free energy is used by enzyme to lower the activation energy of reaction and it provides specificity to reaction.

Chemical Reaction Pathway

Making reactions go faster

• Biological systems are very sensitive to temperature changes

• Increasing the temperature make molecules move faster

• Enzymes can increase the rate of reactions without increasing the temperature

• They do this by lowering the activation energy • They create a new reaction pathway “a short cut”

An enzyme controlled pathway

Most Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions

Substrate binding site consists of an indentation or cleft on the surface of an enzyme. This cleft is complementary in shape to the substrate (geometric complementarity)

The amino acid residues that form the binding site are arranged to interact specifically with the substrate in an attractive manner (electronic complementarity)

Two Models

• Lock and Key • Induced Fit

The Lock and Key Hypothesis

• Proposed by Emil Fisher in 1894• the active site exists “pre-formed” in the enzyme prior to

interaction with the substrate• Fit between the substrate and the active site of the enzyme is

exact , like a key fits into a lock very precisely• The key is analogous to the enzyme and the substrate

analogous to the lock • Temporary structure called the enzyme-substrate complex

formed • Products have a different shape from the substrate • Once formed, they are released from the active site, leaving it

free to become attached to another substrate

The Lock and Key Hypothesis

Enzyme may be used again

Enzyme-substrate complex

E

S

P

E

E

P

Reaction coordinate

The Lock and Key Hypothesis

• This explains enzyme specificity

• This explains the loss of activity when enzymes denature

The Induced Fit Hypothesis

• Some proteins can change their shape (conformation)

• When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation

• The active site is then moulded into a precise conformation

• Making the chemical environment suitable for the reaction

• The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

This model was proposed by Daniel Koshland in 1958It requires the active site to be floppy and substrate to be rigid

Chemical Specificity1. Group Specificity

• Enzymes may act on several different, though closely related substrates

• They catalyse reaction involving a particular group (eg: ALDH)

• ALDH catalyses the oxidation of variety of alcohols

• HK: assist the transfer of PO4 from ATP to several different hexose sugars

2. Absolute specificity

• Enzyme acting only on one particular substrate

• Eg: Glucokinase catalyses the transfer of PO4 from ATP to glucose and to no other sugars

(other egs: urease, arginase, catalase)

3. Steriochemical specificity/ Geometric specificity

• Substrate chemically identical with different arrangement of atoms in in 3D space

• Only one of the isomers undergo reaction by a particular enzyme

L-amino acid KetoacidL-Amino acid Oxidase

Trypsin acts only on polypeptide containing L-amino acids, not those containing of D-amino acidsEnzymes of glucose metabolism are specific for D-glucose

Yeast Alcohol Dehydrogenase (YADH) oxidises ethanol to aldehydesYADH acts on Methanol at 25 fold slowerYADH acts on Propanol 2.5 fold slowerNADPH (differ in a PO4 at 2’ from NADH) does not bind to YADH

• Glycerol Kinase phosphorylates glycerol to Glycerol-3-P

• If phosphorylation occurs at C1, the product is D-glycerol -3-P

• If phosphorylation occurs at C3, the product is L-glycerol -3-P

• The enzyme always produces only L-isomer• Identical chemical groups in a substrate become

different after binding at the microenvironment of the active site of the enzyme (1948, A.G. Ogston)

Enzyme Binding Sites

• Active Site = Binding Site + Catalytic Site

• Regulatory Site: a second binding site, occupation of which by an effector or regulatory molecule, can affect the active site and thus alter the efficiency of catalysis – improve or inhibit

Identification and Characterization of Active

Site

• Structure: size, shape, charges, etc.

• Composition: identify amino acids involved in binding and catalysis.

Binding or Positioning Site(Trypsin)

NH CH C NH

O

N C

complementary binding or posit ioning site

"SPECI FI CI TY"_

+

arginine or lysine

"long + side chain"

H2O

Binding or Positioning Site(Chymotrypsin)

NH CH C NH

O

N C

"aromatic side chain"

"SPECI FI CI TY"

complementary binding or posit ioning site

phenylalaninetyrosinetryptophan

Hydrophobic Pocket

H2O

O

Catalytic Site(e.g. Chymotrypsin)

NH CH C NH

O

N C

catalytic sitecomplementary

"CATALYSI S"

H2O

O

Probing the Structure of the Active Site

Model Substrates

Model Substrates(Chymotrypsin)

H2O(ROH)

NH CH CN

R

NH

O

C

acyl transfer to H2Oaromaticside chain

peptide bond

Peptide Chain?

All Good Substrates!

H3N CH C NH

O

C

R

NH CH CH3N

R

NH2

O

(or -OCH3)

or

H3N CH C

R

NH2

O

(or -OCH3)

or

a-amino group?

Good Substrate!

H2C C NH2

O

R (OCH3)

Side Chain Substitutions

Good Substrates

Cyclohexyl t-butyl-

CH3

CH3

CH3

ConclusionBulky Hydrophobic Binding Site

CH C X

O

Y

"Hydrophobic Acyl Group Transferase"

= hydrophobic posit ioning group

X,Y = various

Probing the Structure of the Active Site

Competitive Inhibitors

Arginase

H2N

C

NH

(CH2)3

CH COOH3N

NH2H2O

NH3

(CH2)3

CH COOH3N

H2N

C

O

NH2

+

+

-+

ureaornithinearginine

+ -

+

Good Competitive Inhibitors

NH3

NH

NH3

(CH2)3

CH COOH3N

NH3

(CH2)4

CH COOH3N

O

(CH2)2

CH COOH3N

CH

NH2

-+

(

ornithine

(+

-

++

+

-

(

canavaninelysine

+

Poor Competitive Inhibitors

All Three Charged Groups are Important

NH3

(CH2)3

CH2H3N

NH3

(CH2)3

H2C COO

CH3

(CH2)3

CH COOH3N+ -

++

+ -

a-aminovaleric acid putrescine

(l,4-diaminobutane)4-aminovaler ic acid

ConclusionActive Site Structure of Arginase

+-

-bindingsite

catalytic site

Identifying Active Site Amino Acid Residues

Covalent Inactivation

Diisopropyl Phosphofluoridate

Inactivates Chymotrypsin by forming a 1:1 covalent adduct to Serine195.

Iodoacetic acid inactivates Ribonuclease by reacting with His12 and His119.

CH O P O CH

CH3

CH3

CH3

CH3

F

O

Affinity Labeling(General Approach)

Positioning Group

Reactive Group

YBinding Site

+

X

X

Affinity Labeling(Tosyl-L-phenylalanine chloromethylketone)

Inactivates Chymotrypsin by forming a 1:1 covalent adduct to Histidine57

O S O

NH

CHCH2

CH3

ReactiveGroup

PositioningGroup

O

C

O

CH2 Cl

Trapping of Enzyme-Bound Intermediate(Chymotrypsin)

Implicates Ser195 in catalytic mechanism.

CT CH2 OH O2N O O C CH3

O

Ser195

O2N

CT CH2 O C CH3

O

O

"acyl" enzyme stable at pH 3

p-nitrophenylacetate

+

O–

p-nitrophenol

Mechanism

http://en.wikipedia.org/wiki/Image:Induced_fit_diagram.svg