enzymes
DESCRIPTION
ENZYME CLASSIFICATION, MECHANISM OF ACTION, SPECIFICITY & ACTIVE SITE.TRANSCRIPT
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
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