This document provides information about enzymes including their history, characteristics, classification, and mechanisms of action. Some key points:
- Enzymes are organic biocatalysts that accelerate chemical reactions by lowering the activation energy. They are not consumed by the reactions they catalyze.
- The term "enzyme" was first used in the 19th century to describe digestion processes. Important early discoveries included identifying enzymes responsible for starch digestion and fermentation.
- Enzymes are usually proteins but can also be RNA. They are highly specific and act as catalysts by lowering the activation energy of reactions through transition state stabilization.
- The International Union of Biochemistry and Molecular Biology (IUBMB)
2. • 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
3. 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.
4. 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
Rockefeller Institute for
Ithaca, NY, USA
Medical Research
Princeton, NJ, USA
Rockefeller Institute for
Medical Research
Princeton, NJ, USA
1887-1955 1891-1987 1904-1971
6. 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 .
7. • Definition:
• Defined as organic biocatalysts synthesized by living cells. They
8. 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.
9.
10. • 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.
11. 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.
12. 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
13. 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
14. 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.
15. • 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
stereospecificity, since they are specialized in the
interconversion of isomers
16. 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.
17. 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
18. • 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.
19. • 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
20. 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
21. • 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
23. 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
24. • 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
26. 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
27. • 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,
28. 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
29. • 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
30. 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'
31. • 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
32. 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
33. • 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
35. 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.
37. Enzyme Nomenclature and Classification
EC Classification
Class
Subclass
Sub-subclass
Serial number
38. Classification
• Based on where enzyme activity occurs –
• Exoenzymes - Digestive enzymes (pepsin, sucrase)
• Endoenzymes- endopeptidases
39. 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.
40. 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
41. • 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
44. 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
45. 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 .
46. • 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.
47. • 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.
48. • 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.
49.
50. • 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.
54. 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”
55. • 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.
56. • 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
62. 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.
63. • Active site of unbound enzyme is complementary in shape to
substrate
64. 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.
65. • Active site forms a shape complementary to substrate only after
it is bound
66. 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.
67. 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
68. 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
69. 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
70. 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
71. 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.
72. 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
73. • Exothermic (exergonic) reactions:
• Energy is liberated in these reactions. E.g. urease
Urea NH3 + CO2 + energy
• Endothermic (endergonic) reactions:
• Energy is consumed in these reactions e.g. glucokinase
Glucose + ATP Glucose 6-phosphate + ADP
74. 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
75. 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.
76. • 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.
77. • 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.
78. • 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
