Enzymes

Definition, Classification- IUBMB, Factors affecting

Enzyme activity, Enzyme kinetics, Inhibition of
activity, Regulation of activity, Measurement of
Enzyme activity, Isoenzymes, Enzymology in medicine

MBBSI, 024
Arun Pandeya
 Definition
• biological catalysts that increase the velocity of
chemical reactions
• no change in overall process
• protein in nature. [except- Ribozymes]
• having the suffix –‘ase’ (exception- pepsin, trypsin, etc.)
• more than 3000 enzymes
Energy of Activation
(EA)
• Enzymes lower EA
• amount of energy
required by reacting
molecules to
undergo the
chemical reactions.

Enzyme catalyzed reactions have lower EA. But, no

difference in net energy between two reactions.
 Nomenclature
1. Recommended name:
• Most of the enzymes are named by adding the suffix-
ase on the substrate of the reaction, eg.
• Maltase: changes Maltose into Glc and Glc
• Urease: converts urea into NH3 & CO2
• Pyruvate carboxylase

• Trivial names: gives no hints on the enzymic reactions.

– eg. pepsin and trypsin, etc.
2. Systemic name: six major classes-
 Mechanism of Enzyme Catalysis
• Enzyme binds its substrate on its active site to
form E-S complex and lastly the product.

E+S ⇄ ES → E+ P
 Enzyme Substrate Binding
Lock-and-Key Model (Emil Fischer)

Active site of the unbound enzyme is complementary

in shape to the substrate
 Enzyme Substrate Binding
Induced-Fit Model (Koshland's Induced Fit Theory)

• enzyme changes shape on substrate binding.

• active site forms a shape complementary to the
substrate only after the substrate has been bound.
 Properties of Enzyme
• Enzymes are biological catalysts and are protein in nature
• Enzyme molecules contain active site, which bind the
substrate to form product.
• Efficiency- Enzyme catalyzed reactions are million times
faster than uncatalyzed reactions.
[turn over number= 102–104s-1 (substrate transformed/sec]
• Specificity- Enzymes are highly specific catalyzing only one
type of chemical reactions.
• Regulations- Enzyme activity can be regulated, i.e.
Enzymes can be activated or inhibited.
 Enzyme Classification: Enzymes can be classified into six
major classes.
S.no. Classes of Examples
Enzyme
1 Oxidoreductase Acyl CoA dehydrogenase, HMG
CoA reductase
2 Transferase Hexokinase, Aminotransferase
3 Hydrolase Digestive enzymes (Amylase,
Lipase)
4 Lyase HMG CoA Lyase, Aldolase
5 Isomerase Phosphoglucose Isomerase, Triose
phosphate isomerase
6 Ligase Glutamine synthetase, Pyruvate
carboxylase
 Enzyme Classification

1. Oxidoreductases:
catalyze the oxidation of
one substrate with
simultaneous reduction of
another substrate.
e.g. Acyl CoAdehydrogenase
Glyceraldehyde-3-P
dehydrogenase
2. Transferases:
transfer a functional
group (e.g. a
phosphate) from
one molecule
(donor) to another
(acceptor).
3. Hydrolases: catalyze the hydrolysis of a chemical
bond by adding water.
eg. All digestive enzymes (Amylase, Lipase and
Trypsin) etc.
4. Lyases:
• catalyze the breaking
of various chemical
bonds by means
other than
hydrolysis and
oxidation.
• eg. Aldolase, HMG
CoA Lyase
5. Isomerases:
• catalyze the
interconversion of
isomers.
• e.g. Triose phosphate
Isomerase,
Phosphoglucose
isomerase.
6. Ligases: catalyze the joining of two molecules with the
involvement of ATP (ATP dependent condensation of
molecules.eg. Pyruvate Carboxylase, Glutamine
Synthetase).
Factors affecting
enzyme activity

The contact
between enzyme
and substrate
determine enzyme
activity
The important factors that influence the velocity
of the enzyme reaction are
1. Concentration of enzyme
2. Concentration of substrate
3. Effect of temperature
4. Effect of pH
5. Effect of product concentration
6. Effect of activators
 1. Concentration of enzyme:

• Rate or velocity (V) of a

reaction is directly
proportional to the enzyme
concentration, when
sufficient substrate is
present.

• This property is used in determining the level of particular

enzymes in plasma.
 2. Effect of concentration of substrate
on increasing the substrate concentration, the velocity
of the enzyme reaction gradually increases within the
limited range of substrate levels.
 3. Effect of temperature
• velocity of reaction
increases with increase in
temperature up to a
maximum & then declines
• The temp at which velocity
of enzyme is maximum is
known as optimum
temperature.
Temperature coefficient (Q10): the factor by which the rate of
reaction is increased when temperature rises in 10°C
 4. Effect of pH
Each enzyme has an
optimum pH at which the
velocity is maximum.
Below and above this pH,
the enzyme activity is
much lower and at
extreme pH, the enzymes
totally inactive.
 5. Effect of activators

• some of the enzymes require certain cofactors

(inorganic ions such as Mg2+, Zn2+, etc) and some
Co-enzymes (such as vitamins) for their activity.
• eg. Vitamin C (ascorbic acid) is the activator of 7-α-
hydroxylase which leads to increased degradation
of cholesterol forming bile salts.
Thus, vitamin C is the activator of the enzyme.
 5. Effect of product concentration

• Accumulation of products generally decreases the

enzyme velocity.
• eg. Accumulation of bile salts inhibits 7-α-
hydroxylase which leads to decreased synthesis of
bile salts.
 Terminologies
• Holoenzyme: functional enzyme having protein part
(Apoenzyme) and non-protein (Cofactor or Coenzyme).

• Coenzyme: small, additional factor which assists enzyme

activity. These are the organic compound and mainly are
derived from B group of vitamins.
• eg. Vitamin C is the coenzyme for 7-α-Hydroxylase

• Cofactor: these assist enzyme activity and are derived

from minerals. These are metal ions such as Zn, Cu etc.
• eg. Cu is the cofactor for Tyrosine hydroxylase
Holoenzyme Apoenzyme + Cofactor
Functional enzyme Protein Non- Protein

If the non-protein part is tightly (covalent) bound to

apoenzyme, it is known as prosthetic group

Small additional factor which assist

enzyme activity, k/a Co-factor

Coenzyme Cofactor
If these are organic If these are inorganic
compound (vitamin metal ions (mineral
derived) derived)
 Coenzymes from B group of Vitamins
Example of Coenzymes Vitamins

Thiamine Pyrophosphate (TPP) Thiamine (B1)

Flavin mononucleotide (FMN or FAD) Riboflavin (B2)

Nicotinamide adenine dinucleotide Niacin (B3)

(NAD+ or NADP+)
(Coenzyme A or CoA) Pantothenic acid
Tetrahydrofolate (THF) Folic acid
 Enzyme kinetics:
Study the rate of enzyme catalyzed reactions
• rate of reaction is assessed by the rate of change of
substrate to product per unit time

• Knowledge of the enzyme catalysis is a prerequisite for

the design of inhibitors (drugs) against a certain enzyme
 Michaelis-Menten Model
• Enzyme reversibly combines with its substrate to
form an ES complex that subsequently yields
product
k1 k2
E + S ES P+E
k-1

Where,
E= enzyme; S= substrate; ES= Enzyme substrate complex;
P= product; and k1k-1k2 are the rate constants
 Michaelis-Menten Equation (simplified form)

vmax [S] if [S] >> KM then v = vmax

v =
[S] + KM
vmax
if [S] = KM, then v = 2

Therefore KM can be viewed as the substrate concentration

with half-maximal velocity.
 Michaelis-Menten Equation- Conclusion

• describes how reaction velocity varies with

substrate concentration.

• reflects the affinity of the enzyme for that substrate.

• KM is numerically equal to the substrate

concentration at which the reaction velocity is
equal to 1⁄2 Vmax.
 Km does not vary with the [E]

• Large Km: reflects a low affinity of enzyme for

substrate [↑S is needed to half-saturate the
enzyme].
• Small Km: reflects a high affinity of enzyme for
substrate, [↓S is needed to half-saturate the
enzyme].
Small Km (Km1)= high
affinity. Because a low
concentration of
substrate is needed to
half-saturate the
enzyme-
i.e. to reach a velocity
which is 1⁄2 Vmax
Fig. Effect of substrate concentration
on reaction velocities for two enzymes:
E1 with a small Km &
E2 with a large Km
 Relationship of velocity to [S]

• [S] is much less than Km, the velocity of the reaction

is approximately proportional to the substrate
concentration [first order].

• [S] is much greater than Km, the velocity is constant

and equal to Vmax.
[rate of reaction is then independent of substrate
concentration, and is said to be zero order].
 v Michaelis-Menten plot
vmax
At Low [S], velocity
of reaction is
proportional to [S]
vmax
2

At high [S], velocity is

independent of [S]

[S]
KM
Effect of substrate concentration
on reaction velocity
 Lineweaver-Burk plot
• When v is plotted against [S], it is not always
possible to determine when Vmax has been achieved
• However, if 1/v is plotted versus 1/[S], a straight line
is obtained.
• This, a double-reciprocal plot can be used to
calculate Km and Vmax, as well as to determine the
mechanism of action of enzyme inhibitors.
Lineweaver-Burke
Plot

Michaelis-Menten Equation

Lineweaver-Burke
Equation
 Use of Lineweaver–Burk plot

• widely used to determine Km and Vmax in

enzyme kinetics
• gives a quick, visual impression of the
different forms of enzyme inhibition.
• competitive
• non-competitive
 Inhibition of Enzyme activity

Inhibitor:
Any substance that can diminish the velocity of
an enzyme-catalyzed reaction.
Two types:
i. Irreversible inhibitors &
ii. reversible inhibitors
 Irreversible inhibitor
 Binds tightly (mostly covalently) at or near the active
site of an enzyme, & form a stable complex
 No dissociation of enzyme and I & enzyme is
permanently inactivated. (or slowly reactivated- hrs or
days for reversal)
• eg. Aspirin covalently modifies the enzyme cyclo-
oxygenase, thus, reduces the synthesis of inflammatory
signals
• Penicillin covalently binds at active site of transpeptidase
and modifies it thereby preventing the synthesis of bacterial
cell walls
Inhibitor may or may not resemble the substrate
 Suicide inhibition
• Irreversible inhibition of enzyme by a substrate
analogue that form reactive species upon enzyme
catalyzed reaction

• eg. Allopurinol, the suicide inhibitor of xanthine

oxidase
 Reversible inhibitor
• The inhibitor forms a loose, dissociable complex (EI)
with the enzyme
• Catalytic activity of EI complex is lower than that of
the enzyme alone.
• Hence, substrate transformation decreases after
addition of the inhibitor.
• Reversible inhibitor may be:
i. Competitive
ii. Non-competitive
iii. Uncompetetive
 Competitive inhibition
1. inhibitor resembles
structurally with the actual
substrate.
2. inhibitor binds reversibly
to the substrate binding
site & competes with the
substrate for that site
eg.
• Inhibition of HMG CoA
reductase by statins
• Inhibition of the succinate
dehydrogenase by
malonate
 Role of HMG CoA Reductase

HMG CoA (6C) Statin

HMG CoA reductase Rate Limiting

Mevalonate (6C)

Cholesterol
Competitive inhibition: Effect on Vmax and on Km

Effect on Vmax:
• The effect of a competitive inhibitor is reversed by
increasing [S].
• At a sufficiently high substrate concentration, the
reaction velocity reaches the ‘Vmax’

Effect on Km
• in the presence of a competitive inhibitor, more
substrate is needed to achieve 1⁄2 Vmax.
 Competitive inhibition Vmax is the same in the
presence of a
competitive inhibitor

Km is increased in
presence of
competitive inhibitor
↑[S]- velocity becomes Vmax
↑[S]- required to achieve Vmax/2
Km- increased by competitive inhibitor
Noncompetitive inhibitor
• A noncompetitive inhibitor can bind
to both free enzyme or ES complex

• Inhibitor binds at site distinct from

active site altering the shape of the
enzyme such that its catalytic activity
is reduced or lost.

non-competitive inhibition can often be reversed by exhaustive

dialysis of the inhibited enzyme
 Noncompetitive inhibitor

• The binding of the heavy metal (Pb) on the enzyme

Ferrochelatase

• Certain insecticides bind covalently on the

acetylcholinesterase, thus, show the neurotoxic
effect
 Regulation of Enzyme Activity
• Enzyme activity can be regulated by
i. Allosteric effectors
ii. Covalent modification
iii. Altered rates of enzyme synthesis or
degradation
 Allosteric Effectors
• bind noncovalently to the enzyme
• can alter the affinity of the enzyme for its substrate or
modify the catalytic activity of the enzyme, or both.
• Effectors my be- Positive or Negative
• Effectors are of two types:
– Homotropic & heterotropic effectors

• Act on the committed step of the metabolic pathway.

 Homotropic effectors

• the substrate itself serves as an effector

• Presence of a substrate molecule at one site on the

enzyme enhances the catalytic properties of the
substrate-binding sites (co-operativity).

• Activation of Aspartate transcarbamoylase by ATP

(pyrimidine biosynthesis).
 Heterotropic effectors
• The effector may be different
from the substrate

• eg. Inhibition of HMG CoA

Reductase by Cholesterol
(product inhibition)
• eg. Inhibition of PFK-1 by citrate
Regulation of enzymes by • by the addition or removal of
covalent modification phosphate groups to/from
the enzyme.
• Phosphorylation and
dephosphorylation is done by
• Protein Kinase
• Protein Phosphatase

• Eg. Glycogen phosphorylase is

active in Phosphorylated form.
Glycogen synthase is active in
dephosphorylated form
 Induction and repression of enzyme synthesis
• cells can also regulate the amount of enzyme
by either increasing (induction) or decreasing
(repression) the enzyme synthesis, leading to
an alteration in the total population of active
sites. eg.
Increase in blood Glucose level

Increased release of Insulin from

pancreatic β-cell

increase in the synthesis of enzymes

involved in glucose metabolism
Enzymes In Clinical Diagnosis
• Enzymology in medicine
• Measurement of Enzyme activity
• Isoenzymes
 Enzymology in Medicine
• Some enzymes which are present in blood/plasma of
normal individuals at a very low levels, they have no any
known function in plasma are known as non-functional
plasma enzymes.
• Presence of these enzymes in plasma is due to cell
turnover.
• However, increased levels of such enzymes in plasma
suggest an increased rate of tissue destruction, thus can
provide diagnostic information.
•Normal level of intracellular enzymes in plasma: Indicates
Normal cell turn over.
•Increased plasma levels of these enzymes: indicate cell
damage, thus provides diagnostic importance.
 Sources of enzymes
• Enzymes are generally synthesized by cells and are
released into circulation.
• eg. ALT- synthesized by liver cells.
AST- synthesized by cardiac cells.
Amylase- synthesized by pancreas and
salivary gland.
Trypsin and Chymotrypsin- by pancreas.
 Clinical significance of Enzymes
Enxym Normal Tissue source Diagnostic importance
es range (U/L)
ALT 8-40 Liver, cardiac , skeletal Hepatic diseases
muscle,
AST 8-40 Cardiac tissues, Cardiac disease, hemolysis,
erythrocytes, Liver hepatic carcinoma
CK 15-100; 10- Muscle, Heart, Brain AMI, Muscular dystrophy
80
LDH 180-360 U/L Heart, RBC, Muscle AMI
ALP 40-125 Bone, Liver, Biliary cells Bone disease, Hepato-biliary
obstruction
GGT 6-45; 5-30 Liver, Biliary cells Hepato-biliary obstruction,
alcoholics
Amyla 28-100 IU/L Pancreas, Parotid gland Acute pancreatitis, parotitis
se
Lipase <40 U/L Pancreas Acute pancreatitis
 Isoenzymes

• Different forms of the same enzymes catalyze similar

type of reactions but present on different tissue
locations.
• Differ in amino acid sequence, hence, differ in physical
properties

Example:
• Isoenzymes of Creatine Kinase (CK):- CK1, CK2 and CK3
• Isoenzymes of Lactate Dehydrogenase (LDH ):- LDH1-
LDH5
Isoenzymes of CK and their normal ranges

1. CK-BB or CK1(brain type)

2. CK-MB or CK2 (hybrid/ cardiac type)
3. CK-MM or CK3 (muscle type)

• Cardiac tissues contain significant quantities of

CK-MB. So injury to cardiac tissues result in
elevation of CK-MB.
Iso-enzyme Tissue Disorders
LDH-1 (HHHH) Heart, RBC MI

LDH-2 (HHHM) RBC, Heart Hemolytic anemia

LDH-3 (HHMM) Lungs, Pancreas Pulmonary

embolism,
pancreatitis
LDH-4 (HMMM) Liver Hepatic injury

LDH-5 (MMMM) Skeletal muscle Skeletal muscle

injury