Enzyme Catalysis

Enzyme Catalysis

Prof. Dr. Supartono, Prof. Dr. Supartono, M.SM.S

PostgraduateSemarang University State

Objective

To understand how enzymes work at the molecular

level.

Ultimately requires total structure determination, but can learn much through biochemical

analysis.

To Be Explained

• Specificity– For specific substrates– Amino acids residues involved

• Catalysis– Amino acids involved– Specific role(s)

• Regulation

24/04/23 3Supartono

EnzymesEnzymes• Enzymes are catalysts. They increase the speed of a chemical reaction without themselves undergoing any permanent chemical change.

• Enzymes are neither used up in the reaction, nor do they appear as reaction products.

24/04/23 4Supartono

Discovery of Enzymes• 1825 Jon Jakob Berzelius discovered 1825 Jon Jakob Berzelius discovered

the catalytic effect of enzymes.the catalytic effect of enzymes.• 1926 James Sumner isolated the 1926 James Sumner isolated the

first enzyme in pure form.first enzyme in pure form.• 1947 Northrup and Stanley 1947 Northrup and Stanley

together with Sumner were together with Sumner were awarded the Nobel prize for the awarded the Nobel prize for the isolation of the enzyme isolation of the enzyme pepsinpepsin..

Berzelius

Sumner

NorthrupStanley24/04/23 5Supartono

Enzyme Characteristics• High molecular weight High molecular weight proteins proteins with masses with masses ranging from 10,000 to ranging from 10,000 to as much as 2,000,000 as much as 2,000,000 grams per molegrams per mole

• Substrate specific Substrate specific catalystscatalysts

• Highly efficienHighly efficien, , increasing reaction increasing reaction rates by a factor as rates by a factor as high as 10high as 1088

24/04/23 6Supartono

Enzyme Nomenclature• The earliest enzymes that were discovered have common names:

i.e. Pepsin, Renin, Trypsin, Pancreatin

• The enzyme name for most other enzymes ends in “ase”

• The enzyme name indicates the substrate acted upon and the type of reaction that it catalyzes24/04/23 7Supartono

Enzyme NamesExamples of Enzyme Names• Glutamic Oxaloacetic Transaminase (GOT)

• L-aspartate: 2-oxoglutarate aminotransferase.

Enzyme names tend to be long and complicated. They are often abbreviated with acronyms24/04/23 8Supartono

Enzyme Mechanisms Enzymes lower the activation energy for reactions and shorten the path from reactants to products

24/04/23 9Supartono

Enzyme Mechanisms The basic enzyme reaction can be represented as follows:

E + S ES E + PEnzyme Substrate Enzyme substrate Enzyme Product(s) complex

The enzyme binds with the substrate to form the Enzyme-Substrate Complex. Then the substrate is released as the product(s).24/04/23 10Supartono

Enzyme MechanismsDiagram of the action of the enzyme sucrase on sucrose.

E+SES E+P

24/04/23 11Supartono

Enzyme Specificity• The action of an enzyme depends primarily on the tertiary and quaternary structure of the protein that constitutes the enzyme.

• The part of the enzyme structure that acts on the substrate is called the active site.

• The active site is a groove or pocket in the enzyme structure where the substrate can bind.24/04/23 12Supartono

Cofactors• Cofactors are other compounds or ions that enzymes require before their catalytic activity can occur.

• The protein portion of the enzyme is referred to as the apoenzyme.

• The enzyme plus the cofactor is known as a holoenzyme.


CofactorsCofactors may be one of three types1. Coenzyme: A non protein organic

substance that is loosely attached to the enzyme

2. Prosthetic Group: A non protein organic substance that is firmly attached to the enzyme

3. Metal ion activators: K+, Fe2+, Fe3+, Cu2+, Co2+, Zn2+, Mn2+, Mg2+, Ca2+, or Mo2+,


Types of Cofactors

• Enzymes have varying degrees of specificity.

• One cofactor may serve many different enzymes. 1524/04/23 15Supartono

Types of Cofactors

• Enzymes have varying degrees of specificity.

• One cofactor may serve many different enzymes.24/04/23 16Supartono

Enzymes and Cofactors


Factors Affecting Enzyme Activity

Enzymes and Reaction Rates

Factors that influence reaction rates of Enzyme catalyzed reactions include1.Enzyme and substrate concentrations

2.Temperature3.pH


Enzymes and Reaction Rates• At low concentrations,

an increase in substrate concentration increases the rate because there are many active sites available to be occupied

• At high substrate concentrations the reaction rate levels off because most of the active sites are occupied


Substrate concentration• The maximum velocity of a reaction is reached when the active sites are almost continuously filled.

• Increased substrate concentration after this point will not increase the rate.

• Vmax is the maximum reaction rate24/04/23 21Supartono

Substrate concentration• Vmax is the maximum reaction rate

• The Michaelis-Menton constant , Km is the substrate concentration when the rate is ½ Vmax

• Km for a particular enzyme with a particular substrate is always the same


Effect of Temperature• Higher temperature increases the number of effective collisions and therefore increases the rate of a reaction.

• Above a certain temperature, the rate begins to decline because the enzyme protein begins to denature


Effect of pH• Each enzyme has an optimal pH at which it is most efficient

• A change in pH can alter the ionization of the R groups of the amino acids.

• When the charges on the amino acids change, hydrogen bonding within the protein molecule change and the molecule changes shape.

• The new shape may not be effective.Pepsin is most efficient

at pH2.5-3 while Trypsin is efficient at a much higher pH


pH


Effects of pH on Enzyme Activity

• Binding of substrate to enzyme• Ionization state of “catalytic” amino acid residue side chains

• Ionization of substrate• Variation in protein structure


Temperature

RelativeActivity

ba

Temperature


Inhibitors

• Covalent– Reversible– Irreversible

• Non-covalent: reversible


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.


Active Site

Binding and Catalysis

General Characteristics

• Three dimensional entity• Occupies small part of enzyme volume

• Substrates bound by multiple weak interactions

• Clefts or crevices• Specificity depends on precise arrangement of atoms in active site


Models

• Lock and Key Model: the active site exists “pre-formed” in the enzyme prior to interaction with the substrate.

• Induced Fit Model: the enzyme undergoes a conformational change upon initial association with the substrate and this leads to formation of the active site.


Enzyme Mechanics

• An enzyme-substrate complex forms when the enzyme’s active site binds with the substrate like a key fitting a lock.

• The shape of the enzyme must match the shape of the substrate.

• Enzymes are therefore very specific; they will only function correctly if the shape of the substrate matches the active site.


Induced Fit Theory


Induced Fit Theory• The substrate molecule normally does not fit exactly in the active site.

• This induces a change in the enzymes conformation (shape) to make a closer fit.

• In reactions that involve breaking bonds, the inexact fit puts stress on certain bonds of the substrate.

• This lowers the amount of energy needed to break them.24/04/23 35Supartono

Induced Fit Theory• The enzyme does not actually form a chemical bond with the substrate. After the reaction, the products are released and the enzyme returns to its normal shape.

• Because the enzyme does not form chemical bonds with the substrate, it remains unchanged.

• The enzyme molecule can be reused repeatedly

• Only a small amount of enzyme is needed

36


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 NHO

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 NHO

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 NHO

N C

catalyt ic sitecomplementary

"CATALYSI S"

H2O

O


Probing the Structure of the Active Site

Model Substrates

Model Substrates(Chymotrypsin)

H2O(ROH)

NH CH CN

R

NHO

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


-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 XO

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)3CH2H3N

NH3

(CH2)3H2C COO

CH3

(CH2)3CH 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

+

XX


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


Catalytic Mechanisms

Mechanisms of Catalysis

• Acid-base catalysis• Covalent catalysis• Metal ion catalysis• Proximity and orientation effects• Preferential binding (stabilization) of the transition state


Acid-Base Catalysis

Keto-Enol Tautomerization

R C CH3

O

R C

OH

CH2

Ketone Enol

Uncatalyzed Reaction


General Acid Catalysis


General Base Catalysis


General Acids

COOH NH3

CH2 OH

CH2 OH

SH

HN NH

CH2


General Bases

COO NH2

SHN N:

CH2


Figure 11-10

Ribonuclease A

N

N

O

O

OH

O

O

CH2

O

PO O

O

N

N

N

N

O

NH2

OH

CH2OPO

O

O

PO O

O

Adenosine

UridineRibonuclease A


Figure 11-10 part 1

Mechanism of RNase A


Figure 11-10 part 2

Mechanism of RNase A


Covalent Catalysis(Principle)

Slow

H2O + A–B ——> AOH + BH

A-B + E-H ——> E-A + BH

E-A + H2O ——> A-OH + E-H

Fast


Covalent Catalysis(Chymotrypsin)

CT CH2 OH O2N OO C CH3

O

CT CH2 O C CH3

O

CT CH2 OH

Ser195

O OO2N

CT CH2 O C CH3

O

HO C CH3

O

"rate limiting"++ H2O

+

f ast+

fast

NOTE: New Reaction Pathway24/04/23 69Supartono

Metal Ion Catalysis

• Metalloenzymes: contain tightly bound metal ions for catalytic activity

• Metal-activated enzymes: loosely bound metal ions from solution

• Charge stabilization• Water ionization• Charge shielding24/04/23 70Supartono

Metalloenzymes

• Catalytically essential (tightly bound): Fe2+, Fe3+, Cu2+, Mn2+, and Co2+

• Structural metal ions: Na+, K+, and Ca2+

• Both: Mg2+ and Zn2+


Proximity and Orientation Effects

Rate of a reaction depends

on:

• Number of collisions• Energy of molecules• Orientation of molecules• Reaction pathway (transition state)


Proximity

V = k[A][B]

[A] and [B] = ~13M on enzyme surface


Biomolecular Reaction of Imidazole with p-Nitrophenylacetate

(Intermolecular)


Intramolecular Reaction of Imidazole with p-Nitrophenylacetate

(Intramolecular)

Intramolecular Rate = 24x Intermolecular Rate24/04/23 75Supartono

Orientation

A BBA

C


Figure 11-14

Geometry of an SN2 Reaction


Stabilize Transition State

HO

R

Br

Y

R

R

Br

Y

R

Z

_

"strain" "stabilized"

HO C

R

XX

CR


Steric Strain in Organic Reactions

Reaction Rate: R=CH3 is 315x vs R=H24/04/23 79Supartono

Figure 11-15

Effect of PreferentialTransition State Binding


Transition State Analogs

Powerful Enzyme Inhibitors

Proline Racemase(planar transition state)


Transition State Analogs of Proline

Binding = 160x versus Proline24/04/23 83Supartono

Serine Proteases

ChymotrypsinTrypsinElastase

etc.

Kinetic Analysis of Chymotrypsin(Hydrolysis of p-nitrophenylacetate)


Mechanism of Chymotrypsin

CT CH2 OH O2N O C CH3

O

CT CH2 O C CH3

O

CT CH2 OH

OO2N

CT CH2 O C CH3

O

HO C CH3

O


+fast+

fast

p-nitrophenolat

e


Identification of the Catalytic Residues

(Reaction of Chymotrypsin with DIPF)

Special Reactivity of Serine195


Identification of the Catalytic Residues

(Reaction of Chymotrypsin with TPCK)

Affinity Labeling24/04/23 88Supartono

X-Ray Structure of Bovine Trypsin(Ribbon Diagram)


Figure 11-26

Active Site Residues of Chymotrypsin(Catalytic Triad)


Figure 11-29 part 1

Catalytic Mechanism of the Serine Proteases

Catalytic Triad


Figure 11-29 part 2



Figure 11-29 part 3



Figure 11-29 part 4



Figure 11-29 part 5



Figure 11-30a

Transition State Stabilization in the Serine

Proteases


Figure 11-30b

Transition State Stabilization in the Serine

Proteases


Mechanism of Chymotrypsin

CT CH2 OH O2N O C CH3

O

CT CH2 O C CH3

O

CT CH2 OH

OO2N

CT CH2 O C CH3

O

HO C CH3

O


+fast+

fast

p-nitrophenolat

e


Enzyme Catalysis

Documents

Transcript of Enzyme Catalysis