Enzymes

Gandham. Rajeev

• Life is short and thus has to be catalyzed.

• Self replication and catalysis are believed to be the

two fundamental conditions for life to be evolved

Enzymes

HISTORY:

• Late 1700 – 1800 - Digestion of starch → sugar

extracts in plants and saliva.

• Meat digestion by secretions in stomach were

identified, but mechanism is unknown.

• In 19th century - Fermentation of Sugar → alcohol in

yeast, studied by Louis Pasteur

• In 1878, German physiologist Wilhelm Kühne first

used the term enzyme, Greek "in living", to describe

this process.

          The Nobel Prize in Chemistry 1946

“ For his discovery that enzymes can be crystallized"

“For their preparation of enzymes & virus proteins in a pure form"

 

                                               

James Batcheller Sumner

John Howard Northrop

Wendell Meredith Stanley

1/2 of the prize 1/4 of the prize 1/4 of the prize

Cornell University Ithaca, NY, USA

Rockefeller Institute for Medical Research Princeton, NJ, USA

Rockefeller Institute for Medical Research Princeton, NJ, USA

1887-1955 1891-1987 1904-1971

Leonor Michaelis(1875-1949)

German

Maud Menten(1879-1960)

Canadian

Enzymes

• Organic bio catalysts - increase the rates of chemical Reactions.

• Accelerate reaction rate by a factor upto 106 or

more • Not consumed / altered by the reactions they

catalyze.• Highly powerful catalytic activity.• Highly Specific in their action• Thermolabile, colloidal in nature • Most of the enzymes are Proteins in nature.• Typical enzyme -Globular protein (62 – 2,500 A.A`s),

M.wt - 12,000 to over 1 million .

• Definition:

• Defined as organic biocatalysts synthesized by living

cells. They are protein in nature (exception - RNA

acting as ribozyme), colloidal and thermolabile in

character, and specific in their action.

• Enzyme catalysis is very rapid; usually 1 molecule of

an enzyme can act upon about 1000 molecules of

the substrate per minute.

• Lack of enzymes will lead to block in metabolic

pathways causing inborn errors of metabolism.

Characteristics of Enzymes

• Almost all enzymes are proteins.

• Enzymes follow the physical and chemical

reactions of proteins.

• They are heat labile.

• They are water-soluble.

• They can be precipitated by protein precipitating

reagents (ammonium sulfate or trichloroacetic

acid).

• They contain 16% weight as nitrogen.

• Ribozymes - RNAs with catalytic activity

• Play role in gene expression rather than metabolism.

• Site - in Cytoplasm, on a cell organelle, membrane

bound, extracellular – interstitial or vascular space.

• In enzymatic reactions - Substrates - the molecules

at the beginning of the process and Products - the

enzyme converts them into different molecules at

the end.

Biomedical Importance

• They determine the patterns of chemical

transformations.

• They mediate the transformation of one form of

energy into another.

• Deficiencies: In the quantity or catalytic activity of

key enzymes - genetic /nutritional deficits, or toxins.

• Imbalances in enzyme activity - pharmacologic

agents to inhibit specific enzymes.

• LIFE IS IMPOSSIBLE WITHOUT ENZYMES.

Naming of Enzymes

• According to the reaction they carry out.

• Suffix - ase is added to the name of the substrate

(e.g., lactase is the enzyme that cleaves lactose) or

the type of reaction

• e.g., DNA polymerase forms DNA polymers).

• Systematic names – based on IUBMB - EC.

• International Union of biochemistry and molecular

biology form system for nomenclature and

classification

Specificity of enzymes

• Enzymes are highly specific in their action

• Specificity is a characteristic property of the

active site

• Types of enzyme specificity:

• Stereospecificity

• Reaction specificity

• Substrate specificity

Stereospecificity or optical specificity

• Stereoisomers are the compounds which have the

same molecular formula, but differ in their structural

configuration

• The enzymes act only on one isomer and, therefore,

exhibit stereospecificity

• L-amino acid oxidase and D-amino acid oxidase act

on L- and D-amino acids respectively.

• Hexokinase acts on D-hexoses

• Glucokinase on D-glucose

• Amylase acts on α-glycosidic linkages

• Cellulase cleaves β-glycosidic bonds

• The class of enzymes belonging to isomerases

do not exhibit stereospecificity, since they are

specialized in the interconversion of isomers

Reaction specificity

• The same substrate can undergo different types of

reactions, each catalysed by a separate enzyme and

this is referred to as reaction specificity.

• An amino acid can undergo transamination, oxidative

deamination, decarboxylation, racemization etc.

• The enzymes however, are different for each of these

reactions.

Substrate specificity

• Absolute substrate specificity:

• Certain enzymes act only on one substrate e.g.

glucokinase acts on glucose to give glucose 6 - phosphate,

urease cleaves urea to ammonia and carbon dioxide

• Relative substrate specificity:

• Some enzymes act on structurally related substances,

• May be dependent on the specific group or a bond

present.

• The action of trypsin is a good example for group

specificity

• Bond Specificity:

• Most of the proteolytic enzymes are showing group

(bond) specificity.

• E.g. trypsin can hydrolyse peptide bonds formed by

carboxyl groups of arginine or lysine residues in any

proteins

• Group Specificity:

• One enzyme can catalyse the same reaction on a

group of structurally similar compounds,

• E.g. hexokinase can catalyse phosphorylation of

glucose, galactose and mannose.

• IUBMB classification of enzymes

• Based on the reaction they catalyze – grouped into

6 major classes - (OTHLIL)

1. Oxidoreductase

2. Transferase

3. Hydrolase

4. Lyase

5. Isomerase

6. Ligase

1. Oxidoreductases:

• This group of enzymes will catalyse oxidation of

one substrate with simultaneous reduction of

another substrate or co-enzyme.

• Catalyze oxidation/reduction reactions.

• They catalyze the addition of oxygen, transfer of

hydrogen & transfer of electrons.

• AH2 + B → A + BH2

• Subclasses:

• Oxidases & dehydrogenases

• Oxidases

• Oxidases catalyse the transfer of hydrogen or

electrons from donor, using oxygen as hydrogen

acceptor - E.g. cytochrome oxidase

• Dehydrogenases:

• Dehydrogenases catalyse the transfer of hydrogen

(or electrons), but the hydrogen acceptor is a

molecule other than oxygen.

• The hydrogen acceptors are usually NAD or NADP &

FAD or FMN - E.g. LDH

Oxido-reductases

2. Transferases:

• This class of enzymes transfers one group (other than

hydrogen) from the substrate to another substrate.

• Transfer a functional group (e.g. a methyl, alcoholic,

aldehyde, ketone, acyl, sulphur or phosphate group).

• A–X + B → A + B–X

• Subclass:

• Transferases (amino transaminases) - amino group

• Kinases - phosphate group

• Aminotransferases (transaminases):

• Catalyse the transfer of an amino group from one

amino acid to an alpha ketoacid, resulting in the

formation of new amino acid & new ketoacid

• E.g. AST

• Transaminases are clinically important.

• Kinases:

• Catalyse the transfer of phosphate from ATP (or

GTP) to a substrate

• E.g. glucokinase

Transaminases

3. Hydrolases:

• This class of enzymes can hydrolyse ester, ether,

peptide or glycosidic bonds by adding water and

then breaking the bond.

• Catalyze the hydrolysis of various bonds, like C-C, C-

O, C-N, P-O and acid anhydride bonds.

• Phosphatases, Esterases, Peptidases, Lipases

A–B + H2O → A–OH + B–H

• Subclass

• Glycosidases & phosphatases

• Glycosidases catalyse the hydrolysis of glycosidic bonds

• E.g. maltase

• Phosphatases catalyse the removal of phosphate from substrate.

• E.g. glucose 6-phosphatase,

4. Lyases:

• These enzymes can remove groups from substrates

or break bonds by mechanisms other than

hydrolysis to form double bonds and addition of

groups to break double bonds.

• Addition or removal of groups to form double

bonds.

• Catalyze cleavage of C-C, C-O, C-N and other bonds

by elimination -

• Elimination and addition reactions.

• Decarboxylases, Synthases

• A -B + X-Y → AX - BY

• Subclass:

• Lyases & Decarboxylases

• Lyases catalyse the cleavage of C-C bonds.

• E.g. citrate lyase

• Decarboxylases catalyse the release of CO2 from the

substrate such as alpha ketoacids & amino acids.

• E.g. Glutamate decarboxylase

5. Isomerases:

• Catalyze intra-molecular group transfer (transfer

of groups within the same molecule).

• These enzymes can produce optical, geometric or

positional isomers of substrates.

• E.g. Epimerases, Mutases, Racemases,

epimerases, cis-trans isomerases

• Interconversion of isomers.

• A → A'

• Subclass:

• Isomerases & epimerases

• Isomerases catalyse the interconversion of cis-trans

isomers & functional isomers

• E.g. Phosphohexoseisomerase.

• Epimerases catalyse the interconversion of epimers.

• E.g. Phosphopentose epimerase

6. Ligases:

• These enzymes link two substrates together,

usually with the simultaneous hydrolysis of ATP,

(Latin, Ligare = to bind).

• Catalyze formation of C-C, C-S, C-O, or C-N bonds,

by condensation reactions, involving ATP.

• A-OH + B-H A-B

• Subclass:

• Carboxylases & syntheteses

• Carboxylases catalyse the formation of C-C bonds

using CO2 (HCO3) as substrate.

• E.g. Pyruvate carboxylase

• Syntheteses are enzymes that link two molecules

with covalent bonds in ATP dependent reaction.

• E.g. Glutamine synthetase

Ligases

ATP →ADP + Pi

IUBMB - EC numbers

• Each enzyme is described by a sequence of four numbers preceded by "EC"

• First digit represents the class - classifies the enzyme based on its reaction.

• Second digit stands for the subclass - indicates the type of group involved in the reaction.

• Third digit is the sub-subclass or subgroup - indicates substrate on which group acts.

• Fourth digit gives the number of the particular enzyme in the list- indicates - serial number of individual enzyme.

Lactate dehydrogenase (lactate:NAD+ oxidoreductase)

Enzyme Nomenclature and Classification

EC Classification

Class

Subclass

Sub-subclass

Serial number

Classification

• Based on where enzyme activity occurs –

• Exoenzymes - Digestive enzymes (pepsin,

sucrase)

• Endoenzymes- endopeptidases

Classification based on complexity

• Simple, monomeric enzymes

• Digestive enzymes (pepsin, sucrase)

• Multimeric

• >1 protein chain, >1 active site

• Multienzyme complexes

• Aggregates of a number of different enzymes

• All enzymes in complex catalyze series of related

reactions

• E.g. FAS complex, PDH complex. etc.

Co-enzymes

• Enzymes may be simple proteins, or complex

enzymes, containing a non-protein part, called the

prosthetic group.

• The prosthetic group is called the co-enzyme.

• It is heat stable.

• Salient features of co-enzymes:

• The protein part of the enzyme gives the necessary

three dimensional infrastructure for chemical

reaction; but the group is transferred from or

accepted by the co-enzyme

• Essential for the biological activity of the enzyme

• It is a low molecular weight organic substance

• The co-enzymes combine loosely with the enzyme

molecules & separated easily by dialysis

• When the reaction is completed, the co-enzyme is

released from the apo-enzyme, and can bind to

another enzyme molecule

• One molecule of the co-enzyme is able to convert a

large number of substrate molecules with the help

of enzyme

• Most of them are derivatives of B complex vitamin

(Vitamins)

Non – Vitamin Coenzymes

ATP Donates Phosphate, adenosine, AMP moieties

CDP Required in phospholipid synthesis as a carrier of choline, ethanolamine

UDP Carrier of glucose – glycogen synthesis galactose

SAM Methyl group donor

PAPS Sulfate group donor in mucopolysaccharide synthesis

Cofactors

• Enzymes may be simple proteins or Compound.

• Many enzymes require small molecules or metal ions to

participate directly in substrate binding or catalysis.

• Active enzyme / Holoenzyme.

• Polypeptide portion of enzyme (apoenzyme)

• Nonprotein prosthetic group (cofactor)

• They can be -

• inorganic metal ions - cofactors or activators.

• complex organic or metallo-organic – coenzymes

• Cofactors are bound to the enzyme to maintain the

correct configuration of the active site .

• Prosthetic groups:

• Some cofactors bind to the enzyme protein very

tightly (non-covalently or covalently).

e.g – FMN, PLP, Biotin, Cu, Mg, Zn

• Metalloenzymes:

• Enzymes with tightly bound metal ions.

• Some metal ions (Fe2+, Cu2+) participate in redox

reactions.

• Others stabilize either the enzyme or substrate

over the course of the reaction.

• Metal-activated enzymes - Enzymes that require

a metal ion cofactor.

• Apoenzyme + cofactor = Holoenzyme

• A holoenzyme also refers to the assembled form

of a multiple subunit protein.

• Holoenzyme:

• A complete, catalytically active enzyme together

with its bound cofactors.

• Certain Vitamins - act as precursors of coenzymes.

• Coenzymes usually function as transient carriers of

specific functional groups -Substrate Shuttles.

• Coenzyme stabilizes unstable substrates such as

H atoms or hydride ions in the aqueous

environment of the cell.

• Second Substrates - Since coenzymes are chemically

changed as a consequence of enzyme action, they

are also named so.

• Common to many different enzymes - about 700

enzymes are known to use the coenzyme NADH.

• Coenzymes are usually regenerated and their

concentrations maintained at a steady level inside

the cell.

• e.g - NADPH is regenerated through the pentose

phosphate pathway & S-adenosylmethionine by

methionine adenosyltransferase.

Figure 5.3

Mechanism of Enzyme Action

• Catalysis is the prime function of enzymes

• For any chemical reaction to occur, the reactants

have to be in an activated state or transition state.

• Generation of transition state complexes &

formation of products:

• Binding of the substrate to the active site of the

enzyme causes bonding rearrangements that leads

to an intermediate state called “transition-complex”

• This is an activated form of substrate immediately

preceding the formation of products.

• An enzyme speeds a reaction by lowering the

activation energy

• Less energy is needed to convert reactants to

products.

• This allows more molecules to form product.

• Activation free energy (G):

• The energy required to convert substrates from

ground state to transition state.

• Substrates need a large amount of energy to

reach a transition state, which then decays into

products.

• The enzyme stabilizes the transition state,

reducing the energy needed to form products

• The enzyme does not affect the equilibrium

position of the reaction

Enzyme-Substrate Binding

Steps of Enzyme Catalysis

• Formation of enzyme – substrate complex.

• Generation of Transition-state complexes

• Formation of Reaction Products

ES Complex

ES Complex

Theories to explain ES Complex

• Lock and key model or Fischer's template theory

• The active site has a rigid shape.

• Only substrates with the matching shape can fit.

• The substrate is a key that fits the lock of the active site.

• Fails to explain the stabilization of the transition state, action

of allosteric modulators.

• Active site of unbound enzyme is complementary in

shape to substrate

Induced-fit Model

• The active sites of some enzymes assume a shape that is

complementary to that of the transition state only after the

substrate is bound.

• The active site is flexible, not rigid.

• Substrate binding brings conformation changes in active site

– nascent active site

• Enables strong binding site - improves catalysis.

• There is a greater range of substrate specificity.

• Active site forms a shape complementary to

substrate only after it is bound

Substrate strain theory

• As the substrate flexes to fit the active site, bonds in

the substrate are flexed and stressed.

• This causes changes/conversion to product.

• Induced fit and substrate strain combinedly operate

in enzyme action.

Mechanism of enzyme catalysis

• The formation of an enzyme-substrate complex (ES) is very

crucial for the catalysis to occur, and for the product

formation.

• It is estimated that an enzyme catalysed reaction proceeds

106 to 1012 times faster than a non-catalysed reaction

• The enhancement in the rate of the reaction is mainly due to

four processes:

• Acid-base catalysis

• Substrates train

• Covalent catalysis

• Entropy effects

Acid-base catalysis

• Role of acids and bases is quite important in enzymology.

• At the physiological pH, histidine is the most important amino

acid, the protonated form of which functions as an acid and its

corresponding conjugate as a base.

• The other acids are –OH group of tyrosine, -SH group of

cysteine, and e-amino group of lysine.

• The conjugates of these acids and carboxyl ions (COO-)

function as bases.

• Ribonuclease which cleaves phosphodiester bonds in a

pyrimidine loci in RNA is a classical example of the role of acid

and base in the catalysis

Substrate strain

• During the course of strain induction, the energy

level of the substrate is raised, leading to a

transition state.

• The mechanism of lysozyme (an enzyme of tears,

that cleaves β -1,4 glycosidic bonds) action is

believed to be due to a combination of substrates

strain and acid-base catalysis

Covalent catalysis

• In the covalent catalysis, the negatively charged

(nucleophilic) or positively charged (electrophilic)

group is present at the active site of the enzyme.

• This group attacks the substrate that results in the

covalent binding of the substrate to the enzyme.

• In the serine proteases (so named due to the

presence of serine at active site), covalent catalysis

along with acid-base catalysis occur, e.g.

chymotrypsin, trypsin etc

Entropy effect

• Entropy is a term used in thermodynamics.

• It is defined as the extent of disorder in a system

• The enzymes bring about a decrease in the entropy of the

reactants.

• This enables the reactants to come closer to the enzyme

and thus increase the rate of reaction.

• In the actual catalysis of the enzymes, more than one of

the processes acid-base catalysis, substrate strain, covalent

catalysis and entropy are simultaneously operative.

• This will help the substrate (s) to attain a transition state

leading to the formation of products.

Thermodynamics of enzymatic reactions

• The enzyme catalysed reactions may be broadly

grouped into three types based on thermodynamic

(energy) considerations.

• lsothermic reactions:

• The energy exchange between reactants and

products is negligible. e.g. glycogen phosphorylase

Glycogen + Pi Glucose 1-phosphate

• Exothermic (exergonic) reactions:

• Energy is liberated in these reactions. E.g. urease

• Endothermic (endergonic) reactions:

• Energy is consumed in these reactions e.g. glucokinase

Glucose + ATP Glucose 6-phosphate + ADP

Urea NH3 + CO2 + energy

Active site

• The active site (or active centre) of an enzyme

represents as the small region at which the

substrate(s) binds and participates in the catalysis

• Active site is due to tertiary structure of protein.

• Clefts / crevices – provide suitable environment for

reaction

Salient features of active site

• The existence of active site is due to the tertiary

structure of protein resulting in three dimensional

native conformation

• The active site is made up of amino acids (known as

catalytic residues) which are far from each other in

the linear sequence of amino acids (primary

structure of protein).

• For instance, the enzyme lysozyme has 129 amino

acids.

• Lysozyme has 129 amino acids.

• The active site is formed by the contribution of amino

acid residues numbered - 35, 52, 62, 63 and 101.

• Active sites are regarded as clefts or crevices or

pockets occupying a small region in a big enzyme

molecule

• The active site is not rigid in structure and shape.

• It is rather flexible to promote the specific substrate

binding.

• The active site possesses a substrate binding site and

a catalytic site.

• The latter is for the catalysis of the specific reaction.

• The coenzymes or cofactors on which some enzymes

depend are present as a part of the catalytic site.

• The substrate (s) binds at the active site by weak non-

covalent bonds.

• Enzymes are specific in their function due to the

existence of active sites.

• The commonly found amino acids at the active

sites are serine, aspartate, histidine, cysteine,

lysine, arginine, glutamate, tyrosine.

• Among these amino acids, serine is the most

frequently found.

• The substrate (s) binds the enzyme (E) at the

active site to form enzyme-substrate complex (ES)

• The product (P) is released after the catalysis and

the enzyme is available for reuse

Thank you