ENZYMES

DR.M.S.GAYATHRI MD
ASST PROFESSOR
STANLEY MEDICAL COLLEGE

Willy Kunhe (1878)- coined the term enzyme

Edward Buchner (NB:1907) cell free extract of yeast
could catalyze the fermentation of sucrose to ethanol
&named it as Zymase.
James Sumner(NB:1946) crystallize the enzyme urease.
Definition of Enzyme
 colloidal, thermolabile,organic catalysts synthesized
by living cells, but active in both inside & out side
the cell & even in cell-free extracts.
 A protein with catalytic properties due to its power
of specific activation
 Enzymes are proteins and certain class of RNA
(ribozymes) which enhance the rate of a
thermodynamically feasible reaction and are not
permanently altered in the process
Catalysts
 Substance which increases the velocity of the
chemical reaction, but not consumed in the
net chemical reaction or process
The substrate
 The substrate of an enzyme are the reactants
that are activated by the enzyme
 Enzymes are specific to their substrates
 The specificity is determined by the active
site
Characteristics
Enzyme-catalyzed reactions have the
following characteristics in comparison
with the general catalyzed reactions:
 common features:
 unique features:
Common features
 Do not consume themselves: no
changes in quantity and quality before
and after the reactions.
 Do not change the equilibrium points:
only enhance the reaction rates.
 Apply to the thermodynamically
allowable reactions
 Reduce the activation energy
Unique features
 Enzyme-catalyzed reactions have very
high catalytic efficiency.
 Enzymes have a high degree of
specificity for their substrates.
 Enzymatic activities are highly regulated
in response to the external changes.
Molecular Components
 All enzymes are proteins with large Mol.wt,
heat laible,soluble in water, Precipitated by
protein precipitating agents
 Simple Enzymes: only amino acids no non
protein part.
Conjugated Enzymes
Apoenzyme (Protein Part)
H – Large molecular
o wt.
l – All protein
o
+Contains active
–
=
e site
Coenzyme (Non protein Part)
n –Small molecular wt
z –Organic
y or
m Cofactor
e –Inorganic
–Metallic
Molecular Components
 In addition to enzymes, other chemical
species often participate in the catalysis.
 Cofactor: chemical species required by
inactive apoenzymes (protein only) to
convert themselves to active
holoenzymes.
Cofactors
 An additional non-protein molecule that is
needed by some enzymes to help the reaction
Eg:metal ions; small organic molecules
 Tightly bound cofactors are called prosthetic
groups
 Cofactors that are bound and released easily
are called coenzymes
 Many vitamins are coenzymes
 Cofactors

Cofactors

Essential ions Coenzymes

Activator ions Metal ions of Cosubstrates Prosthetic

(loosely bound) metalloenzymes (loosely bound) groups
(tightly bound) (tightly bound)
 Essential ions
 Activator ions: loosely and reversibly bound,
often participate in the binding of substrates.
Eg: Cations : Hexokinase- Mg++, Acetyl CoA
carboxylase-Mn++, 6-P gluconate
dehydrogenase-Mg++ or Mn++ or Ca+,
Arginase- Mn++ or Co++ Anions
: Amylase – Cl-
 Metal ions of metalloenzymes: tightly bound, and
frequently participate directly in catalytic
reactions.
Metallo-enzymes
 Zinc  Carbonic anhydrase, Carboxy
peptidase,alcoholdehydrogenase
 Magnesium  Hexokinase,phospho fructo kinase,enolase,
glucose-6-phosphatase
 Manganese  Phospho gluco mutase, hexokinase, enolase,
glycosyl trasferases
 Copper  Tyrosinase, cytochrome oxidase, lysyl oxidase,
superoxide dismutase.
 Iron  Cytochrome oxidase, catalase, peroxidase,
xanthinoxidase.
 Calcium  Lecithinase, lipase
 Molybdenium  Xanthineoxidase
Function of metal ions

• Transfer electron
• Linkage of S and E ；
• Keep conformation of E-S complex
• Neutralize anion
Organic compounds

 Small size and chemically stable

compounds
 Transferring electrons, protons and other
groups
 Vitamin-like or vitamin-containing
molecule
 Coenzymes
 Low M.Wt organic substance, heat stable
 Essential for biological activity of enzyme
 Loosely bind to apoenzyme. Be able to be separated with dialysis.
Two groups
1.Take part in reactions catalyzed by Oxidoreductases by accepting H+ and
leaving to transfer it to others, or vise versa.
eg: NADP-NADPH+, FAD-FADH2 and FMN-FMNH2
2.Take part in reactions transferring groups other than hydrogen
eg: Vit-B complex
 Metabolite coenzymes: they are synthesized from the common
metabolites.
 several NTP, ATP (most abundant), UDP-glucose
 Vitamin-derived coenzymes: they are derivatives of vitamins, and can
only be obtained from nutrients.
 NAD and NADP+, FAD and FMN, lipid vitamins, …
Common Coenzymes
 Coenzyme Reaction Catalyzed
 Biotin Carboxylation
 Cobalamin (B12) Alkylation transfer
 Coenzyme A Acyl transfers
 Flavin Oxido-Reduction
 Lipoic acid Acyl transfers
 Nicotinamide Oxido-Reduction
 Pyridoxal phosphate Amino group transfers
 Tetrahydrofolate One carbon group
transfers
 Thiamine pyrophosphate Aldehyde transfer
Coenzymes
 Act as group-transfer reagents to
supply active sites with reactive groups
not present on the side chains of amino
acids
 Cosubstrates:
 Prosthetic groups:
Co substrates

 The substrates in nature.

 Their structures are altered for
subsequent reactions.
 Shuttle mobile metabolic groups among
different enzyme-catalyzed reactions.
Prosthetic groups
 Supply the active sites with reactive
groups not present on the side chains
of AA residues.
 Can be either covalently attached to its
apoenzymes or through many non-
covalent interactions.
 Remained bound to the enzyme during
the course of the reaction.
 Molecular Components
 Monomeric Enzymes- one polypeptide chain
Eg: Ribonuclease, DNA Poly-I.
 Oligomeric Enzymes- more than one polypeptide chain
Eg: LDH, Hexokinase, Gly-3-P-dehydrogenase,
Pyruvatekinase Ect.
 Multienzyme complex- different enzyme catalyzing
reaction sites are located at different sites of the same
macromolecule for catalyzing different consecutive
reactions of metabolic pathway
Eg: Fattyacid synthetase, Carbamoyl phosphate
syntetaseII, Pyruvate dehydrogenase
Proenzymes or Zymogen
 Some enzymes are synthesized as larger inactive
precursor forms called proenzymes or zymogens.
 This prevents them from catalyzing reactions in the
cell where they are synthesized
 Activation involves the irreversible hydrolysis of
one or more peptide bonds, resulting in an active
form.
 Precursor proteins or inactive enzymes names have
the prefix “pro” ;like prothrombin, proelastase etc.
or sufix “ogen” like chymotrrypsinogen,
trypsinogen, pepsinogen etc.
Ribozyme
 Until recently, all the enzymes are known to be
proteins.
 Ribonucleic acids also demonstrate the catalytic
ability.
 Ribozymes have the ability to self-cleave.
 They are highly conservative, an indication of the
biological evolution and the primary enzyme.
 Ribozyme cleave RNA phosphodiester bonds at
specific sites in the RNA molecule, serving as
ribonucleases and as peptidyl transferases
Enzyme structure
 Enzymes are
proteins
 They have a
globular shape
 A complex 3-D
structure

Human pancreatic amylase

The active site
 One part of an enzyme,
the active site, is
particularly important
 The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
more easily
Active site
 Each type of enzyme molecule contains a
unique, intricately shaped binding surface
called an active site.
 Active centers look like a cleft or a crevice.
 Active centers are hydrophobic.
 Change in primary, secondary, tertiary or
quaternary structure may alter the three
dimensional shape of the active site & reduces
its binding and catalytic activity.
Lysozyme

Residues (colored ) in the active site come from

different parts of the polypeptide chain .
Two essential groups

The active center has two essential groups in general.

• Binding group: to associate with the reactants to form
an enzyme-substrate complex.
• Catalytic group: to catalyze the reactions and convert
substrates into products.
Catalytic residues are highly conserved. Certain
amino acids, notably cysteine and hydroxylic,
acidic, or basic amino acids, perform key roles in
catalysis
• Essential group in active site: binding group
+catalytic group. Cofactors always be a part of the
active site
 Protein chain

Substrate
molecule
Essential groups
outside the
active center
+ Catalytic group
-
Active center

Binding group
Enzyme-Substrate Complex
 Single-substrate reactions: eg; isomerases E+
S ↔ ES ↔ E + P
 Single-displacement bisubstrate reactions: eg;
hexokinases, hydrolases E+
S1 ↔ ES1 ES1+ S2 ↔
ES1S2 (ternary complex) ↔ EP2 + P1 EP2 ↔ E
+P2
 Double-displacement bisubstrate reactions:
transaminases,transketolase,transaldolase E+
S1 ↔ ES1 ↔ E` + P1 E` + S2 ↔
E`S2 ↔ E` + P2
 Double displacement multisubstrate reactions:
acetyl-CoA carboxylase
Mode of action of Enzymes
 Catalytic activity of Enzyme: they accelerate
reactions at least a million times by reducing the
energy of activation.
Chemical reactions need an initial input of
energy = THE ACTIVATION ENERGY
During this part of the reaction the molecules are
said to be in a transition state.
Reaction pathway
transition state, S
G+ (uncatalyzed)
Free energy

reactants

G for
the reaction
products

Reaction progress
Making reactions go faster
 Increasing the temperature make molecules move
faster
 Biological systems are very sensitive to temperature
changes.
 Enzymes can increase the rate of reactions without
increasing the temperature.
 They do this by lowering the activation energy.
 They create a new reaction pathway “a short cut”
Enzymes speed up metabolic
reactions by lowering energy barriers
 Enzyme speed reactions by lowering
activation energy
 The transition state can be reached at
moderate temperatures.
 Enzymes do not change delta G.
 It speed-up reactions that would occur
eventually.
An enzyme controlled pathway
transition state, S
G+ (uncatalyzed)
Free energy

G+ (catalyzed)

reactants

G for
the reaction

products

Reaction progress
Catalysis: How enzymes work
 Common Mechanisms
Proximity and Strain Effects
 Substrate fits the catalytic site with proper
orientation to catalytic groups. Enzyme conformation
changes to give a strained E-S complex.– Facilitates
attaining the transition state.
 eg lysozyme- enzyme of tears cleaves β 1,4
glycosidic bond due to substrate strain
Electrostatic Effects
 Substrate binding site excludes H2O & lowers the
dielectric constant strengthening electrostatic
interaction between E and S.
 Catalytic Mechanisms
Acid-Base catalysis
-Enzyme side chains act as proton donors and
acceptors. eg-ribonuclease cleaves
phosphodiester bond in pyrimidine loci in RNA
Covalent Catalysis
Powerful nucleophilic or electrophilic side chain
forms an unstable covalent bond to the
substrate. In serine proteases both covalent
&acid base catalysis eg trypsin ,chymotrypsin
Metal Catalysis
 Orients substrates + stabilizes charge in the
transition state + supplies or captures electrons
during the course of the reaction.
 Metal bound at the active site of enzymes can act as
electrophilic catalysts, stabilizing the increased
electron density of negative charge that can develop
during reaction.
 Metal can also provide a nucleophile at neutral pH
by coordinating to a group with ionizable proton
 Example: Alcohol dehydrogenase
Mechanism of Enzyme Action
The Lock and Key Hypothesis or Rigid Template
model of Emil Fisher
 Fit between the substrate and the active site of the enzyme is
exact
 Like a key fits into a lock very precisely
 The key is analogous to the enzyme and the substrate
analogous to the lock.
 Temporary structure called the enzyme-substrate complex
formed
 Products have a different shape from the substrate
 Once formed, they are released from the active site
 Leaving it free to become attached to another substrate
The Lock and Key Hypothesis

S
E
E
E

Enzyme- Enzyme may

substrate be used again
complex P

Reaction coordinate
The Lock and Key Hypothesis
 This explains enzyme specificity
 This explains the loss of activity when
enzymes denature.
 This model explains all mechanisms but do
not explain the changes in the enzyme activity
in the presence of allosteric modulators.
The Induced Fit Hypothesis or Hand
in Glove Model of Koshland
 Some proteins can change their shape (conformation
 Enzymes are flexible and shapes of the active site can be
modified by binding of the substrate.
 When a substrate combines with an enzyme, it induces a
change in the enzyme’s conformation
 The active site is then moulded into a precise conformation
 Making the chemical environment suitable for the reaction
 The bonds of the substrate are stretched to make the reaction
easier (lowers activation energy)
 This can explaincomplex saturation kinetics,competitive
inhibitions & allosteric modulations.
 Induced-fit model

The binding induces conformational changes of both

E and S, forcing them to get a perfect match.
The Induced Fit Hypothesis

Hexokinase (a) without (b) with glucose substrate

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

 This explains the enzymes that can react with a

range of substrates of similar types
Conventional Nomenclature
 Adding the suffix –ase to the name of the substrates
(urease)
 Adding the suffix –ase to a descriptive term for the
reactions they catalyze (glutamate dehydrogenase)
Trivial names
 For historic names or Whimsical names (trypsin,
amylase)
 Being named after their genes (Rec A –recA,
HSP70)
Systematic Nomenclature

 The International Union of Biochemistry and

Molecular Biology (IUBMB) maintains the
classification scheme.
 Categorize in to 6 classes according to the
general class of organic reactions catalyzed
 Assigned a unique number, a systematic
name, a shorter common name to each
enzyme
1.Oxidoreductases
Enzymes that catalyze electron transfer oxidation-reduction
reactions
AH2 + B → A + BH2

Enzymes in this class: dehydrogenases, reductases, oxidases &

peroxidases.
Eg- Alcohol + NAD+ → Aldehyde + NADH + H+
Alcohol dehydrogenase (alcohol-NAD+ oxidoreductase, E.C.
1.1.1.1.)
Lactate dehydrogenase (NAD) , Glu-6-P-
dehydrogenase(NADP), succinate dehydrogenase (FAD),
ferrooxidase, Cytochrome oxidase
L- and D-amino acid oxidase , malate dehydrogenase
 2.Transferases
Catalyzing transfer of groups between donors and acceptors such as
amino, carboxyl, methyl, phosphoryl etc
A-X + B → A + B-X

Enzymes in this class Aminotransferases, kinases

&Transcarboxylase

Eg- Hexose + ATP → Hexose-6-phosphate + ADP

Hexokinase (ATP-D-hexose 6-phosphotransferase,
E.C.2.7.1.1.)

Transaminase
Transmethylases
3.Hydrolases

Catalyzing cleavage of bonds(ester, ether,peptide or

glycosidic ) by addition of water
A-B + H2O → A-OH + B-H

Acetyl choline + H2O → Choline + acetate

Acetyl choline esterase or hydrolase
Lipase (triacylglycerol acyl
hydrolase, E.C. 3.1.1.3.)
All digestive enzymes like amylase, pepsin,trypsin etc
Acid and alkaline phosphatases
Urease
 4.Lysases
Catalyzing lysis of a substrate and generating a double bond
(nonhydrolytic, and non-oxidative reactions)
C-X C
| → ║ +XY
C-Y C

Eg: FructoseI,6-bisphosphate ↔ DHAP +Glyceraldehyde-3-P

Aldolase (ketose 1-phosphate aldehyde lyase, E.C. 4.1.2.7.)
Fumarase (C-O)lyases,
Histidine decarboxylase (C-C) lyases , HMG CoA lyase
 5.Isomerases

Catalyzing intramolecular rearrangement (structural or

geometric changes )in a molecule. They are called
racemases, epimerases, isomerases or mutases of
optical or geometric isomers
A → A’
Glyceraldehyde-3-P → DHAP
Triose phosphate isomerase ( D-glyceraldehyde 3-
phosphate ketoisomerase, E.C. 5.3.1.1.)
Retinol isomerase
Phosphohexose isomerase
 6.Ligases

Catalyzing synthetic reactions forming a covalent bond

(C-O, C-S, C-N, C-C ) at the expense of a high
energy bond of ATP
A + B +ATP → A-B+ ADP +Pi

Glutamate + NH3 + ATP → Glutamine +ADP + Pi

Glutamine synthetase (L-glutamate ammonia ligase,
E.C. 6.3.1.2.)
Acetyl CoA carboxylase
Pyruvate carboxylase
Enzyme specificity

An enzyme can bind with only such

substrates as have steric conformations
and groups fitting with the three-
dimensional conformations of the active
site and its specific amino acid side
chains. so it can bind to and act on only a
limited number of substrates fulfilling such
specifications. This is known as the
substrate specificity of enzymes.
Enzyme specificity
Unlike conventional catalysts, enzymes demonstrate the
ability to distinguish different substrates. There are
four types of substrate specificities.
 Absolute specificity
 Group specificity
 Reaction specificity
 Stereo specificity
Absolute specificity
 Enzymes can recognize only one type of
substrate and implement their catalytic
functions
 Glucose Glucose-6-Phosphate
 Lactose Glucose + Galactose
 Urea Ammonia +Co2
Group Specificity
 Group of compounds may serve as substrate for an
enzyme.
Hexokinase
Phosphatases hydrolyses any organic phosphate as
long as the ester bond and phosphate group are there.
Proteinases(Peptidases)are proteolytic
enzymes like Exopeptidase
(hydrolysing terminal peptide bond) eg:
Carboxypeptidase, Aminopeptidase. Endopeptidase
(hydrolysing centrally located peptide bond) eg:
pepsin, trypsin and chymotrypsin
Reaction specificity
Enzymes are specific in their action. Almost
only one enzyme catalyzes a given specific
reaction.

Pyruvate dehydrogenase Lactate dehydrogenase

Acetyl-CoA Pyruvate
Lactate

Pyruvate carboxylase Transaminase

Oxaloacetate Alanine

Each reaction is catalyzed by a separate enzyme, which catalyzes only that reaction
Stereo specificity
Many enzymes possess diff. types of stereo specificity.
They act only on a specific stereoisomer of the
substrate and not on its other stereoisomer.

H H

C C
OH COOH
H3C COOH H3C OH

B B C
A C A
Stereo specificity
 D-L stereospecificity:
Eg; Lactate dehydrogenase can recognize only the L-form but
not the D-form lactate. L-aminoacid oxidase & D-aminoacid
oxidase act only on L-aminoacids & D-aminoacids respectively,
similarly D-glucose oxidase acts only on D-glucose.
 Geometric or cis-trans specificity:
Many enzymes, acting on substrates with double bonds in the
carbon chain, exhibit cis-trans specificity Eg; Aconitase of
mitochondria can act only on cis-aconitic acid & not on trans-
aconitic acid; fumarase of mitochondria acts only on fumaric
acid (trans isomer), but not on maleic acid (cis isomer)
Salivary
amylase acts only on alfa-1,4 glycoside linkage