Enzymes

Bettelheim, Brown, Campbell and Farrell

Chapter 23

CO20

Enzymes

• Catalyst: Speeds up rate of reaction but does not change equilibrium. The catalyst itself is not changed.

• Enzymes are protein molecules which catalyze a chemical reaction

• Enzymes catalyze specific reactions on specific compounds (single isomers)

• Business part of enzyme is the “active site”

• Active site binds the substrate (compound which undergoes a reaction)

Enzyme Names

• Generally come from the name of the reaction that enzyme catalyzes.

• Frequently end in “ase”Name of substrate - ending

+ aseLactose –ose +ase = lactase

Alcohol dehydrogenase

Categories of Enzymes

• Oxidoreductases—catalyze redox reaction

• Transferases—catalyze transfer of functional group to a different molecule– Kinase—transfer of phosphate group– Transaminase—transfer of amino group

• Hydrolases—hydrolysis reactions (add water and break bond

Categories of Enzymes

• Lyases—add group to double bond OR remove group to make double bond

• Isomerases—rearrange to make isomer

• Ligases—join two molecules– May involve several kinds of bonds:

• C-C C-S C-O C-N

How do enzymes catalyze a reaction?

Enzymes lower activation energy for a reaction

They do not change the equilibrium constant, only the RATE of the reaction

Terminology

• Substrate: compound on which enzyme acts

• Active site: part of enzyme where substrate binds

• Activation: process of making an inactive enzyme active

• Inhibition: process that makes an enzyme less active or inactive– Competitive inhibition– Noncompetitive inhibition

Enzyme Activity

• Enzyme activity:Enzyme activity: a measure of the reaction rate for an enzyme

• Rate of enzyme-catalyzed reaction is affected by– enzyme concentration– substrate concentration – temperature– pH

Enzyme Concentration

• Enzyme concentration

Enzyme Activity

Substrate Concentration

Rate will level off as sites on enzyme are filled up

• Initial rate of reaction will double when you double amount of substrate

• Rate increases to a maximum velocity

when all of the active sites on an enzyme are full

Vmax

Effect of Temperature

Enzyme most active at optimum temperature

Effect of Temperature on Enzyme Rate

• Uncatalyzed reaction rates increase as temperature increases

• Enzymes have temperature optimum, at which the enzyme has its highest rate– Generally about 37oC for many enzymes– Above the temperature optimum, enzyme

rates fall – At high temperatures, enzyme is denatured

Fig. 20.10 Uncatalyzed

Reaction

Catalyzed

Reaction

Effect of pH

Enzyme most active at optimum pH

Effect of pH on Enzyme Rate

• Enzymes have different reaction rates at different pH’s

• pH Optimum: pH at which rate is highest– Near pH 7 for many enzymes– Some have optima at very high or low pH

• At pH higher or lower than optimum, rate falls off

• At extreme pH, enzyme will be denatured

Formation of Enzyme-Substrate Complex

E + S ↔ ES ↔ ES* ↔ EP ↔ E + P

Enzyme Enzyme Transition Enzyme Enzyme

+ substrate state product +

Substrate complex complex Product

Overall Reaction: S → P

Enzyme-Sutbstrate.mov

Enzyme Mechanism

• Substrate fits into the active site and then undergoes a reaction

• Enzyme-substrate complex formed

• Enzyme-substrate complex is an intermediate species

• Initial binding of substrate relatively fast

• Conversion of substrate to product (and release of product) is slower

• This is “Rate Limiting Step”

Characteristics of Active Site

• Site where substrate binds to enzyme• Generally have groups that extend into the

active site to help catalyze the reaction– Often histidine

• Substrate “fits” into site.• Substrate held by weak, noncovalent

interactions in “binding site”• Site very specific—only substrate that fits

into site will undergo reaction

Enzyme Specificity

• Enzyme specificity is the ability of an enzyme to bind only one (or a very few) substrates and thus catalyze only one reaction

Fig. 20.12

Levels of Specificity

• Absolute: One substrate only

• Group: Similar compounds (hexoses)

• Linkage: Recognize bond (linkage) types

• Stereochemical: D- or L- isomer

Two Models for Enzyme Activity

• Lock and Key Model– Emil Fischer 1894– Substrate fits into “rigid” active site just as a key fits into

a lock

• Induced Fit Model– Daniel Koshland 1958– Enzyme modifies its shape to accommodate the

substrate

induced fit.movEnzyme-Sutbstrate.mov

Induced Fit Model

Lock and Key Model

– Both the lock-and-key model and the induced-fit model emphasize the shape of the active site

– the chemistry of the active site is the most important• just five amino acids participate in the active sites in more

than 65% of the enzymes studies to date

– these five are His > Cys > Asp > Arg > Glu

– four of these amino acids have either acidic or basic side chains; the fifth has a sulfhydryl group (-SH)

Look at enzyme-substrate complex again

Focus on steps in forming transition state and product

Formation of Enzyme-Substrate Complex

E + S ↔ ES ↔ ES* ↔ EP ↔ E + P

Enzyme Enzyme Transition Enzyme Enzyme

+ substrate state product +

Substrate complex complex Product

Overall Reaction: S → P

Enzyme-Sutbstrate.mov

How transition state helps reaction to proceed more rapidly

• Put “stress” on bond in substrate

• Bring reactants closer together

• Put reactants into correct orientation

• Provide different pH environment in active site

Stress Bond

Correct Orientation

Regulation of Enzyme Activity

Rate will level off as sites on enzyme are filled up

Enzyme Inhibitors

• Bind to enzymes and eliminate or reduce catalytic activity.

Types of Inhibitors

• Irreversible Inhibitors

• Reversible, Competitive Inhibitors – Structural Analogs

• Reversible, Noncompetitive Inhibitors

Competitive Inhibition

– the induced-fit model explains competitive inhibition

– the inhibitor fits into the active site, preventing the substrate from entering

competitive inhibition.mov

Noncompetitive Inhibition

• Noncompetitive inhibitors– Inhibitor binds

elsewhere on enzyme and changes the active site so substrate can’t attach

noncompetitive inhibition.mov

Mechanism of Action– we can distinguish between competitive and

noncompetitive inhibition by the enzyme kinetics in the absence and presence of the inhibitor

Enzyme Regulation

Feedback Inhibition of enzyme pathway

A → B → C → D → E → F

If enough F is present, it can bind to an enzyme earlier in sequence and inactivate it. This stops synthesis of all subsequent products.

Enzyme Regulation

• Feedback control:Feedback control: an enzyme-regulation process where the product of a series of enzyme-catalyzed reactions inhibits an earlier reaction in a sequence

– the inhibition may be competitive or noncompetitive

A B C DE1 E2 E3

feedback inhibition

feedback control.mov

Activating Enzymes

• Apoenzyme: Protein portion of enzyme

• Cofactor: Non-protein prosthetic group– Examples: Metal ions such as Zn2+, Mg2+

• Coenzyme: Organic prosthetic group– Examples: Heme, Vitamins

• Holoenzyme: Active enzyme

Cofactor binds and changes active site

Activating enzymes

• Coenzyme: Bind temporarily to catalytic site to help catalyze reaction (often have vitamin component)

• Coenzyme binds to apoenzyme first.• Substrate binds second

• Both product and coenzyme are released after reaction

1

2

3

4

Examples of Vitamin Coenzymes

Regulation of Enzyme Activity

• Allosteric Enzymes:– More than one binding site– Shape of active site is changed by binding of

molecules to another part of enzyme– Regulator molecules bind to regulatory site

Positive Allosterism: Changes to active form

Negative Allosterism: Changes to inactive form

Fig. 20.11

Regulation of Enzyme Activity

• Zymogen (Proenzyme): Enzyme originally made in inactive form. Part of it must be removed before it is active.– Trypsinogen—activated by cleaving off 6 aa

• Protein Modification: Group can be bound or removed to activate or inactivate an enzyme. Easily reversed.– Phosphorylation of enzyme turns it on/off

Examples of Zymogens

Isozymes

• Isozymes (isoenzymes) catalyze the same reaction

• Different forms in different tissues

• May be inhibited or turned on by different molecules

• Frequently have one isozyme active normally

• Other isozyme can be induced when needed