• Enzymes are:

• Proteins (exception: few RNA molecules), colloidal in

nature
• Catalyze chemical reactions (catalytic site)
• Increase the rate of reactions without being changed
in the process
• Can be denatured and hydrolyzed into component
amino acids
• Enzyme acts on substrate to yield product
• Nomenclature:
• Recommended name: have the suffix “-ase” attached to
the substrate of the reaction
• For example: sucrase, lactase, urease, lipase
• Named by their discovers for a broad function:
• E.g. pepsin: digestion of food
• Lysozyme: lyse bacterial cell wall
• Trypsin: to wear down
• Systemic name (developed by IUBMB)
• divided into six major classes , each with numerous
subgroups
• Each enzyme is assigned a four-part classification
number and a systematic name, which identifies the
reaction it catalyzes.
For example:
ATP + D-glucose → ADP + D-glucose-6-phosphate

• formal systematic name is ATP : glucose

phosphotransferase , which indicates that it catalyzes
the transfer of a phosphoryl group from ATP to
glucose.
• Its Enzyme Commission number (E.C. number) is 2.7.1.1.
• first number (2) denotes the class name (transferase)
• second number (7), the subclass (phosphotransferase)
• third number (1), a phosphotransferase with a hydroxyl
group as acceptor
• fourth number (1), D-glucose as the phosphoryl group
acceptor
• For many enzymes, a trivial name is more commonly used
—in this case Hexokinase.
Important terminology

• Holoenzyme: Apoenzyme (protein part)+ non-protein part

• Coenzymes: A complex organic molecule (vitamin B-
complex derivates. E.g. Coenzyme A, Pyridoxal phosphate,
Tetrahydrofolate, FAD, NAD)
• Cofactors: bind in a transient & dissociable manner to the
enzyme (most commonly metal ions)metal activated
enzyme
• Prosthetic groups: coenzyme or metal ion that is very
tightly/covalently bound to the enzyme. Metals are the
most common prosthetic groups metalloenzymes
Examples:

• Fe2+ (Cytochrome oxidase, catalase, peroxidase)

• Mg2+ (Hexokinase , glucose 6-phosphatase,pyruvate
kinase)or
• Zn2+ (Carbonic anhydrase, alcohol dehydrogenase).
PROPERTIES OF ENZYMES

A. Active sites:
• A special pocket or cleft where substrate binds and forms
enzyme–substrate (ES) complex.
• Contains amino acid side chains that create a three-
dimensional surface complementary to the substrate.
• ES is converted to an enzyme–product (EP) complex that
subsequently dissociates to enzyme and product.
Substrate binding model to Enzyme

• Fischer's lock and key model

• Daniel Koshland's induced fit model
B. Catalytic efficiency
 103 to 108 times faster than uncatalyzed reactions
 Number of molecules of substrate converted to product
per enzyme molecule per second is called the turnover
number, or kcat
C. Specificity
• Enzymes are highly specific, interacting with one or a
few substrates and catalyzing only one type of chemical
reaction.
• Enzymes are also stereospecific catalysts and typically
catalyze reactions of only one stereoisomer of a given
compound—for example, D- but not L-sugars, L- but not
D-amino acids
D. Regulation
Enzymes can be activated or inhibited, so that the rate of
product formation responds to the needs of the cell.
 Allosteric regulation: effectors molecules bind to allosteric
site
 Homotropic effectors: when substrate itself is an
effector, act as a positive effector, sigmoidal curve
 Heterotropic effectors: effector is different from the
substrate. e.g. Feedback inhibition
 covalent modification
 By phosphorylation and dephosphorylation
 Glycogen phosphorylase is active in phosphorylated
state
 Glycogen synthase is active in dephosphorylated state
 Induction and repression of enzyme synthesis
• Slow process (hrs to days) compared to previous
mechanisms (seconds to minutes)
• Induction: increase enzyme synthesis
• Repression: decrease enzyme synthesis
E. Location within the cell
• Many enzymes are localized in specific organelles within
the cell.
• Such compartmentalization serves to isolate the reaction
substrate or product from other competing reactions.
• provides a favorable environment for the reaction, and
organizes the thousands of enzymes present in the cell
into purposeful pathways.
HOW ENZYMES WORK
FACTORS AFFECTING REACTION VELOCITY

• Substrate concentration
• as substrate concentration is increased, Vi increases
until it reaches a maximum value Vmax
• When further increases in substrate concentration do
not further increase vi, the enzyme is said to be
“saturated” with substrate
Representation of an enzyme at low (A), at high (C), and
at a substrate concentration equal to Km (B)
• Temperature
• reaction velocity increases with T until a peak velocity is
reached (optimum T)
• Further elevation of T results in a decrease in reaction
velocity because enzymes get denatured
• Enzymes from humans exhibit stability up to 45–55 °C
• Q10, (temperature coefficient) is the factor by which the
rate of a biologic process increases for a 10°C increase in
T
• For T over which enzymes are stable, the rates of most
biologic processes typically double for a 10 °C rise in T
(Q10 = 2)
• pH
• Most intracellular enzymes exhibit optimal activity at pH
values between 5 and 9.
• For enzymes whose mechanism involves acid-base
catalysis, the residues involved must be in the
appropriate state of protonation
MICHAELIS-MENTEN EQUATION

• Rate equation for one-substrate enzyme-catalyzed reaction

• Quantitative relationship between the V0 (initial velocity) Vmax
(maximum velocity), initial substrate concentration [S], and
Km (Michaelis constant)
Michaelis-Menten equation
Significance of KM & Vmax Values
KM and Vmax, can be readily derived from rates of catalysis
measured at a variety of substrate concentrations
KM is the concentration of substrate at which half the active
sites are filled
Vmax reveals the turnover number of an enzyme, which is the
number of substrate molecules converted into product by
an enzyme molecule in a unit time when the enzyme is fully
saturated with substrate
 Lineweaver-Burk plot
• can be used to calculate Km and Vmax, as well as to
determine the mechanism of action of enzyme inhibitors.

FIG: Double-reciprocal or Lineweaver-Burk plot

Enzyme Inhibition

• Any substance that can diminish the velocity of an

enzyme-catalyzed reaction is called an inhibitor
• Irreversible inhibition: Covalent
inhibitors/Mechanism based inhibitors
• Reversible inhibition: Non-covalent/ transition-state
analogs
• Competitive
• Non-competitive
• Competitive inhibition:
• inhibitor binds reversibly to the active site (substrate
analogs)
• Effect on Vmax: Unchanged
• Effect on Km: Increases
• E.g. statin drugs (simvastatin, atorvastatin) inhibit HMG-
CoA reductase of cholesterol synthesis
• Malonate inhibits Succinate dehydrogenase of Krebs
cycle
• Noncompetitive inhibition:
• inhibitor and substrate bind at different sites on the
enzyme.
• Bear little or no resemblance to substrate
• Effect on Vmax: decreases
• Effect on Km: Unchanged
• e.g. Heavy metal toxicity is caused by tight binding of a
metal such as Hg, Pb, Al, or Fe, to a functional group(-
SH) in an enzyme.
• Pb inhibits Ferrochelatase of heme synthesis
• Insecticides inhibit acetylcholinesterase
• Irreversible Inhibitors:
• Inhibitors act irreversibly by chemically modifying the enzyme (making or
breaking covalent bonds with aminoacyl residues essential for substrate
binding, catalysis, or maintenance of the enzyme’s functional conformation)
• Enzymes are poisoned
• Iodoacetate inhibit glyceraldehyde 3-phosphate dehydrogenase
• Diisopropyl fluorophosphate (DFP) irreversibly binds with enzymes
containing serine at the active site, e.g. serine proteases, acetylcholine
esterase.
• penicillin antibiotics act as irreversible inhibitors of serine – containing
enzymes, and block the bacterial cell wall synthesis. (Glycopeptidyl
transferase)
• Cyanide inhibits cytochrome oxidase (binds to iron atom) of electron
transport chain.
• Fluoride inhibits enolase (by removing manganese), and thus glycolysis)
MECHANISM-BASED INHIBITORS (Suicide Inhibition):
• Original inhibitor (the structural analogue/competitive
inhibitor) is converted to a more potent form by the same
enzyme that ought to be inhibited
• Allopurinol, an inhibitor of xanthine oxidase, gets converted
to alloxanthine, a more effective inhibitor of this enzyme.
• 5-fluorouracil gets converted to fluorodeoxyuridylate which
inhibits the enzyme thymidylate synthase, and thus
nucleotide synthesis
Isoenzymes and Clinical Enzymology
 Isoenzymes
• Isoenzymes are multiple forms of an enzyme that
possess the ability to catalyze the enzyme’s
characteristic reaction but differ in structure because
they are encoded by distinct structural genes
• Different isoenzymes may arise from different tissues
their specific detection gives clues to the site of pathology
• Various isoenzymes of an enzyme can differ in three
major ways:
- catalytic properties
- physical properties (e.g. heat
stability/electrophoresis/resistance to chemical)
- biochemical properties such as amino acid
composition and immunological reactivity
Genetic origins of enzyme variants

• True isoenzymes are due to the existence of more

than one gene locus coding for the structure of the
enzyme
• Genes at the different loci have undergone differential
modifications during the course of evolution so that
the enzymes coded by them have no longer identical
structure
• Isoenzymes genes are not necessarily closely linked on
one chromosome; they are often located on different
chromosomes
• E.g. Genes that code for human salivary and pancreatic
amylases both are located on chromosome 1, whereas
• Genes that code for mitochondrial and cytoplasmic Malate
dehydrogenase are carried on chromosomes 7 and 2
respectively
• Enzymes of clinical importance that exist as
isoenzymes because of the presence of multiple-gene
loci are:
• Lactate dehydrogenase
• Creatine kinase
• α-amylase
• some forms of alkaline phosphatase
• Allelic variation/Allozymes:
• enzyme forms differ from one individual to another
because of the existence of alternative alleles
• human gene loci subject to allelic variation is considerable
and frequency of occurrence differs from one enzyme to
another
• Placental Alkaline phosphatase and Glucose-6-phosphate
dehydrogenase have high incidence of mutant alleles at
single locus
• When isoenzymes, because of variation at a single
locus, occur with appreciable frequency, the
population is said to be polymorphic with respect
to the isoenzymes in question
• Oligomeric enzymes:
• Enzymes made up of subunits
• Association of different types of subunits in various
combinations gives rise to a range of isoenzymes
• When the subunits are derived from different structural
genes (multiple loci/alleles) hybrid isoenzymes
• E.g. CK-MB, LD-2, LD-3, LD-4
Nongenetic Causes of Multiple Forms of
Enzymes

• Post-translational modifications of enzyme molecules give

rise to multiple forms known as isoforms
• De-amidation: amylase, carbonic anhydrase
• Sulfhydryl oxidation: adenosine deaminase, acid
phosphatase
• Removal of C-terminal amino acid: CK-MM and CK-MB
isoforms
• Variations in carbohydrate side chains (sialic acid): liver
and/or bone ALP
• Aggregation of enzyme: Cholinesterase
• Enzyme-protein complex: Macroamylase
• Distribution of Isoenzymes and Other Multiple
Forms of Enzyme
• Distribution of isoenzymes is not uniform throughout the body,
and wide variations in the activities of different isoenzymes are
found at the organ, cellular, and subcellular levels
• Isoenzyme LD-X or LDc found only in Testes
• Placental alkaline phosphatase is detectable only in placenta
• Mitochondrial and cytoplasmic AST and Malate
dehydrogenase
• Basis for organ specific diagnosis through isoenzyme
measurements
Measurement of serum enzymes
 Enzymes are normally intracellular and LOW
concentration in blood
 Enzyme leakage in the blood indicates cell injury (cell
death, hypoxia, intracellular toxicity)
 Some enzymes are found predominantly in specialized
tissue, e.g. lipase in pancreas
 Widely distributed enzymes, e.g. AST, ALP.
 Isoenzymes or isoforms can be evaluated to enhance
tissue and organ specificity
• Fairly non invasive, possible to do repeated tests
• Enzyme’s diagnostic window:
• Interval of time following an episode of injury during
which plasma concentrations of enzyme are increased,
thereby demonstrating the occurrence of injury
• Early markers and late markers
• Enzyme activity is expressed in International unit
(IU/L)
• corresponds to the amount of enzymes that catalyzes
the conversion of one micromole (mol) of substrate to
product per minute
Information from enzymes measurements in serum

 Presence of disease and site of pathology

 Altered enzyme production: increase or decrease in level
 Etiology /nature of disease: differential diagnosis
 Physiological condition: high level of ALP in growing children
 Enzyme induction: increase level of Y-GT which results from
administrations of drugs (barbiturates/phenytoin/alcohol
intake)
 Ectoenzymes: Y-GT may be eluted from surfaces of liver cells
through accumulation of bile salts without cell damage
 Clearance of enzyme from circulation: α-amylase is excreted
in Urine
 Extent of disease  more damaged cells more
leaked enzymes in blood
 Acute and chronic disease: Use of early indicators and late
indicators
 E.g. High level of cytoplasmic AST indicates acute liver
disease whereas mitochondrial AST is marker of chronic
liver disease
 Time course of disease:
 Start rising after onset of disease, peak level and then back
to normal level
 Half-lives vary from hrs to days (average 6-48hrs)
 Enzymes routinely measured
NAME OF THE ENZYME PRESENT IN

Aspartate Amino transferase (AST) Heart , Liver, RBC, skeletal muscle

Alanine Amino transferase (ALT) Liver

Alkaline Phosphatase (ALP) Liver, Bone, intestine and Placenta

Acid Phosphatase (ACP) Prostate

 glutamyl Transferase ( GT) Liver, pancreas, kidney

Creatine kinase (CK) Skeletal Muscle , heart

Lactate Dehydrogenase (LDH) Heart, liver, muscle, RBC

 - Amylase Pancreas, salivary gland

Lipase Pancreas
LACTATE DEHYDROGENASE (LDH)

 LDH is elevated in myocardial infarction, blood disorders

 It is a tetrameric protein and made of two types of subunits
namely H = Heart, (chromosome 12) M = skeletal muscle
(chromosome 11)
 It exists as 5 different isoenzymes with various combinations
of H and M subunits
Isoenzyme name Composition Present in Elevated in

LDH1 ( H4) Myocardium, RBC Myocardial

infarction

LDH2 (H3M1)
Myocardium, RBC

(H2M2)
LDH3 Kidney, Skeletal muscle

LDH4
(H1M3) Kidney, Skeletal muscle

LDH5 (M4) Skeletal muscle, Liver Skeletal muscle

and liver
diseases
• Serum LDH measurement is relevant in hematology
and oncology
• Leukemia, hemolytic anemia
• Probability of survival in Hodgkin’s disease and non-
Hodgkin’s lymphoma
• Malignant disease with metastases to liver and lymph
nodes
• Germ cell tumors: teratoma, seminoma of testes,
dysgerminoma of ovary
 CREATINE KINASE (CK)

Creatine kinase is a dimer made of 2 monomers

Skeletal muscle contains M subunit (Chromosome
14), Brain contains B subunits (chromosome 19)
Three different isoenzymes are formed
Isoenzyme name Composition Present in Elevated in

Brain damaged
CK-1 BB Brain neonates and
LBW newborns

Myocardium/ Acute
CK-2 MB myocardial
Heart infarction

Skeletal
CK-3 MM muscle, Skeletal muscle
injuries
Myocardium
• Skeletal muscle injuries:
• Duchenne muscular dystrophy
• Rhahbdomyolysis
• Viral myositis, polymyositis
 ALT & AST
 ALT & AST enzymes are the most abundantly present in the liver
 Measurement of these transaminases is useful for the diagnosis
of parenchymal liver diseases
 In most types of liver disease, ALT> AST
 Alcoholic hepatitis, cirrhosis & liver neoplasia, AST> ALT
 In viral hepatitis the enzyme levels are increased 20-50xULN
 Other causes like toxic hepatitis and NAFLD
 ALT increase is specific for liver damage involving hepatocellular
damage
 AST is moderately increased in Muscular dystrophy and acute
myocardial infarction
 ALKALINE PHOSPHATASE (ALP)
 Have maximal activity at a high pH 9.0-10.5
 High levels are seen in liver, bone, placenta & intestine and
useful to assess hepatobiliary and bone diseases
 In hepatobiliary obstruction, hepatocytes lining the biliary ducts
induces the ALP synthesis.
 High levels of ALP is indicative of extrahepatic obstruction rather
than intrahepatic obstruction
 In bones, the enzyme is derived from osteoblasts. Hence
increased in bone diseases like rickets, osteomalacia, neoplastic
diseases with bone metastates and healing fractures
 Physiological condition with high ALP
 Third trimester of pregnancy
 Growing children
  glutamyltransferase ( GT)
 It is involved in amino acid transport across the
membranes
 Found mainly in biliary ducts of the liver, kidney and
pancreas
 Enzyme activity is induced by a number of drugs
(phenytoin/phenobarbital)and alcohol
 -GT increased in liver diseases especially in obstructive
jaundice
 -GT levels are used as a marker of alcohol induced liver
disease and in liver cirrhosis
 AMYLASE
 Digestive enzymes from pancreas (p-type) and salivary
glands (s-type)
 Elevated in acute pancreatitis and salivary gland
inflammation
 It is used as a marker to detect acute pancreatitis &
appendicitis
 Hyperamylasemia also occurs in neoplastic disease
Tumors of the lung and serous & mixed carcinomas 
of the ovary can produce hyperamylasemia 
LIPASE
• Lipase concentration is very high in pancreas
• Serum level of lipase is used to diagnose acute pancreatitis
• Lipase is more specific than amylase in diagnosing
pancreatitis
• Remain elevated longer than amylase
• Help in late diagnosis / delayed presentation
• Three causes of pancreatitis:
• Hypertriglyceridemia
• Obstructive jaundice
• Heavy drinking
 ACID PHOSPHATASE (ACP)

 Have maximal activity at pH 5.0-6.0

 It is present in prostate gland, liver, spleen and RBC
 The main source of ACP is prostate gland and so can be
used as a marker for prostate disease

 Other causes of high ACP:

 High osteoclastic activity; giant cell tumor, marble bone
disease
 Lysosomal storage disease; Gaucher’s disease
 Hairy cell leukemia
Therapeutic use of Enzymes

• Therapeutic enzymes have a broad variety of specific

uses
• Oncolytics
• Thrombolytics
• Anticoagulants
• Replacement of metabolic deficiencies
• Digestive aids
• Metabolic storage disorders
• Prolactazyme: lactose intolerance
• Beta-Lactamase: Penicillin allergy
• Streptokinase: heart attack (clot blusters)
• Asparaginase: acute childhood leukemia
• Collagenase: skin ulcers
• Lysozyme: antibiotic therapy
• Dnase: cystic fibrosis