ENZYME- BIOLOGICAL CATALYST. All enzymes are proteins - with the exception of some RNAs that catalyze their own splicing all enzymes are proteins In general,

ENZYME-ENZYME-BIOLOGICAL BIOLOGICAL

CATALYSTCATALYST

• All enzymes are proteins - with the exception of some RNAs that catalyze their own splicing all enzymes are proteins

• In general, names end with suffix “ase”- tyrosinase (tyrosine), celullase (cellulose), protease (protein), lipase (lipid)

• Enzyme: a biological catalyst• enzymes can increase the rate of a reaction by a

factor of up to 1020 over an uncatalyzed reaction

Enzyme As Catalyst

• Catalysis:

- the process of increasing the rate of chemical reactions

• Catalyst:

- the substance that facilitate in catalysis

• Enzymes are catalysts:- increase the rate of a reaction- not consumed by the reaction- act repeatedly to increase the rate of reaction- enzymes often “specific” – promote only 1 particular reaction, others catalyze a family of similar reactions

cellulase – cellulose as substratehexokinase – any 6 ring monosaccharide -

fructose, glucose

Enzyme As Catalyst

• Higher reaction rates:- the rates of enzymatically catalyzed reactions are 106 to 1020 > than uncatalyzed reaction

• Milder reaction rates:- enzyme catalyzed reactions occur under relatively milder conditions: < 100oC, atmospheric pressure and nearly neutral pH- contrast with chemical catalysis requires high temperature, pressures and extremes pH.

• Greater reaction specificity: - enzyme have greater degree of specificity to their substrates and their products – rarely have side products

General Properties of Enzyme

Enzyme CatalysisActive site - part of enzyme to which the substrate binds and the reaction takes place

Substrate – a reactant in an enzyme-catalyzed reaction

Enzyme-substrate (ES) complex – the intermediate formed when the substrate is bind at the active site of an enzyme

Product

Enzyme Catalysis

GENERAL FORMULA

E = enzyme

S = substrate

P = product

Enzyme catalysis reaction• Physically interact with their substrates to

effect catalysis• E + S ES ES* EP E + P• Where:

- E = enzyme- ES = enzyme/substrate complex- ES* = enzyme/transition state complex- EP = enzyme/product complex- P = product

• Substrate bind to the enzyme’s active sitepocket in the enzyme

• Catalytic site = active site = where reaction takes place

Enzyme catalysis reaction

• E + S ES ES* EP E + P

• 1st step: enzyme binds to substrate molecule to form an enzyme – substrate complex

• E + S ES


Enzyme


• 2nd step: Formation of the transition state complex where the bound substance is neither product nor reactant

• ES ES*, ES≠ES



• 3rd step: Formation of the enzyme – product complex ES* EP



• 4th step: Release of product EP E + P


• Enzyme can only work on one substrate molecule at a time

• Not change during the reaction

• One product is release, enzyme is available to accept another substrate molecule


• Rate of reaction = reaction velocity (V)- the rate of enzyme reaction is measured by the rate of the appearance of products or the rate of disappearance of substrates. - d[P]/dT or d[S]/dTmol product/min or mol substrate/min

• Enzyme activity? 1 unit (U) is the amount of enzyme that catalyses the

reaction of 1 mol of substrate per minute under specified conditions.

Enzyme Catalysis

Enzyme Catalysis • The rate of a reaction depends on

its activation energy, G°‡

• an enzyme provides an alternative pathway with a lower activation energy

• Activation energy – the energy required to start a reaction

• Transition state – the intermediate stage in a reaction in which the old bonds break and new bonds are formed

How enzyme work? • Transition state theory:– The enzyme (E) must approach

the substrate (S), the substrate attach to the active site through noncovalent bond

– Formed the high energy (unstable) ES complex

– In ES complex, the covalent bond in substrate is in the process of breaking while the EP complex is forming.

Enzyme Catalysis - Example

• Consider the reaction

H2O2 H2O + O2

catalase

a) No catalyst,

b) with added Fe3+ salt,

c) with added catalase

• (a) – a/e for the reaction in the absence of a catalyst

• (b) – a/e lowered in the presence of an iron catalyst

• (c) – energy diagram for the catalase-catalyzed breakdown of H2O2

• (d) – energy diagram for the noncatalysed breakdown of H2O2 at elevated temperature

a/e – activation energy

Active site• Has specificity – can discriminate among possible

substrate molecules- others recognize a functional group (group specificity)- only recognize one type of molecule (eg. D vs L isomer)

(absolute specificity)• Relatively small 3D region within the enzyme

- small cleft or crevice on a large protein• Substrates bind in active site by weak non-covalent

interactions (Hydrogen bond, hydrophobic and ionic interaction)

Active site• The interactions hold the substrate in the proper

orientation for most effective catalysis

• The energy derived from these interactions – binding energy

• Binding energy is used, in large part to lower the activation energy and stabilize the transition state complex (ES*)

• Each non-covalent interaction provides energy to stabilize the transition state

Binding Models• Two models have been developed to describe

formation of the enzyme-substrate complex

1.1. Lock-and-key modelLock-and-key model:: substrate binds to that portion of the enzyme with a complementary shape

2.2. Induced fit model:Induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

Enzyme/substrate interaction1.1. Lock and key modelLock and key model

- substrate (key) fits into a perfectly shaped space in the enzyme (lock)

- lots of similarities between the shape of the enzyme and the shape of the substrate

- highly stereospecific

- implies a very RIGID inflexible active site

- site is preformed and RIGID

Enzyme/substrate interaction

2.2. Induced fit modelInduced fit model (hand in glove analogy)

- count the flexibility of proteins

- substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S

- change in protein configuration leads to a near perfect fit of substrate with enzyme

Figure 6.3, pg 148

Campbell & Farrell, Biochemistry, 6th Ed., 2009, Thomson Brooks/Cole

• You need to understand:

1. Factors effecting enzyme reaction rate

2. 6 classes of enzymes

3. Cofactor, coenzymes, holoenzymes, apoenzymes

4. Allosteric enzyme, effectors (positive and negative), heterotropic and homotropic allosterism

5. Isoenzyme and multienzyme

Key words for today

• What influence the enzyme reaction rate?

1. Substrate concentration

2. Temperature

3. pH

4. Enzyme concentration

5. Inhibitor

Characteristics of enzyme reactions

• Substrate Saturation: Increasing the [substrate] increases the rate of reaction (enzyme activity).

– enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time.

– At the saturation point,the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau.


[substrate] = substrate concentration

• Temperature

- very sensitive to temperature changes- low temp, rate of an enzyme-catalysed reaction increases proportionally with increasing temperature


• Effects of Temperature: All enzymes work within a

range of temperature specific to the organism.

Increases in temperature lead to increases in reaction rates - is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates - due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme.


• pH

- enzymes have an optimal pH at which they function properly

- varies to each other but most in the range of pH 6-8


pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.

• Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity.

All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds.


• Enzyme concentration

- the higher the concentration, the greater should be the initial reaction rate – will be lasting as long as substrate present


• Inhibitor

- inhibit enzyme by occupy the active site or bind to other part of enzyme – leading to the change of enzyme shape and eventually the active site

- this will decrease the enzymatic reaction rate


Classification of Enzymes• Have 6 categories

• Each enzyme has an official international name ending with –ase and a classification number

• Number consists in 4 digits (referred to a class and subclass of reaction

Classification of Enzymes

Classification of Enzymes

Table 5.1, pg 136

Boyer, R., Concepts in Biochemistry, 3rd Ed., 2006, John Wiley &Sons

Enzyme Classes: Examples

Class Example Reaction

1

(oxidoreductase)

alcohol dehydrogenase

2

(transferase)

hexokinase glucose + ATP glucose-6-phosphate

+ ADP

3

(hydrolase)

chymotrypsin polypeptide + H2O peptides

CH3CH2OH CH3CH

O

+ NAD+ + NADH + H+

More examples:Refer Table 5.2, pg 136 , Boyer, R., Concepts in Biochemistry, 3rd Ed., 2006, John Wiley &Sons

Enzyme Classes: Examples

Class Example Reaction

4

(lyase)

pyruvate

decarboxylase

5

(isomerases)

alanine

racemase

D-alanine L-alanine

6

(ligases)

pyruvate

carboxylase

CH3CO

CO

O CH3CHO

+ H+ + CO2

CH3CO

CO

O

O CO

CH2CO

CO

O

+ HCO3-

ATP ADP+Pi

_

Enzymes & cofactor• Enzymes require chemical entity in order to function

properly (assists an enzyme in catalytic action)

• Cofactor – nonprotein molecule that assist in an enzyme catalytic reaction

• Coenzyme – smaller organic or organometallic molecule derived from vitamin, weakly bound to enzyme, temporarily associated with enzymes

• Prosthetic group – coenzymes that are covalently or noncovalently tightly bound to enzyme and always present.

Enzymes & cofactor• Holoenzyme – an

enzyme in its complete form including polypeptide(s) and cofactor

• Apoenzyme – enzyme in its polypeptide form without any necessary prosthetic groups or cofactors

Allosteric Enzymes• Allosteric enzymeAllosteric enzyme:: an oligomer whose biological

activity is affected by other substances binding to it• these substances change the enzyme’s activity by

altering the conformation(s) of its 4°structure

• Allosteric effectorAllosteric effector:: a substance that modifies the behavior of an allosteric enzyme; may be an• allosteric inhibitor = negative effectors• allosteric activator = positive effectors

Allosteric enzyme in feedback InhibitionFormation of product inhibits its continued production – feedback inhibition

Allosteric Enzymes (Cont’d)

• The key to allosteric behavior is the existence of multiple forms for the 4°structure of the enzyme

• allosteric effector:allosteric effector: a substance that modifies the 4° structure of an allosteric enzyme

• homotropic effects:homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase

• heterotropic effects:heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP

ATCase = aspartate transcarbomylase

CTP = cytidine triphosphate

Allosteric enzymes• A change in conformational structure at one location

of a multisubunit protein that causes a conformational change at another location on the protein

• Effectors – i) serves as stimulants to enzyme (+ve effectors) = increase catalytic activity – ii) inhibitors (-ve effectors) to enzyme = reduce/inhibit catalytic activity- Act by reversible, noncovalent binding to a site on the enzyme

• Larger and more complex than nonallosteric enzyme• Have 2 or more subunits (oligomeric)• Allosteric enzymes have regulatory sites for binding

of substrates and reaction (catalytic sites)

HOMOTROPIC ALLOSTERISM• Eg. Tetrameric allosteric enzyme

composed of 4 identical subunits• Each subunit has a catalytic site

where substrate/effector will bound and transformed to product

• Once bound to active site, a message will transmitted via conformational changes to an active site on another subunit which makes it easier for a substrate molecule to bind and react at that site

• This type (substrate and effector) are the same is called cooperative or homotropic

HETEROTROPIC ALLOSTERISM• A dimer with nonidentical subunits• Subunit α contain the active site –

catalytic subunit• Subunit β contains the site for

effector binding – regulatory subunit

• Binding of a specific effector molecule to the regulatory site on the β subunit sends a signal via conformational changes to the catalytic site on subunit α

• Substrate and effector different kinds of molecules - heterotropic

Isoenzymes• Enzymes that catalyze the same reaction

(catalytically and structurally similar) but are encoded by different genes

– Glycogen phosphorylase-synthesize in liver, brain and muscle-involves in degradation of glycogen

– Isoenzymes = isoforms

Multienzymes

– A group of noncovalently associated enzymes that catalyze 2 or more sequential steps in metabolic/biochemical pathway

ENZYME- BIOLOGICAL CATALYST. All enzymes are proteins - with the exception of some RNAs that catalyze their own splicing all enzymes are proteins In general,

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