Enzymology
 ASHFAQ
Learning Objective
 Define what are Enzymes
 Describe Properties of Enzymes
 Classify Enzymes
 Discuss Structure of Enzymes
 Justify Clinical Significance of
Enzymes
Enzymes
What are Enzymes
Enzymes
Structure of Enzymes
Most Enzymes Are
Proteins
Enzymes
Structure of Enzymes
Enzymes
RNA enzyme-Ribozymes break the
phosphodiester bonds of RNA in ribosome
Structure of Enzymes
Enzymes
Single subunit
Enzyme
Glucosidase converts maltose
into two molecules of glucose
Structure of Enzymes
Enzymes
Multi-subunit Enzyme
Dimer of HIV
protease
Tetramer of Lactate
dehydrogenase
Structure of Enzymes
Active site
Cleft or Pocket on the surface
provide
I. Binding site
II.
catalytic site
Enzymes
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 which
are far from each other in the linear sequence of
amino acids
Salient features of active site
The enzyme
lysozyme has 129
amino acids.
The active site is
formed by the
contribution of amino
acid residues
numbered 35, 52, 62,
63 and 101.
Salient features of active site
The active site is not rigid but flexible to promote
the specific Substrate binding.
The substrates binds at the active site by weak
non- covalent bonds.
The commonly found amino acids at the active
sites
are serine, aspartate, histidine, cysteine,lysine,
arginine, Glutamate, tyrosine etc.
The hydroxyl group of Ser195 is the
nucleophile
Models for Enzyme substrate complex
A-Enzyme complementary to substrate
Proposed by Emil Fischer
in 1894
Enzymes structurally
complementary to
substrates, fit together like a
lock and key
Every Lock has its own
Key
Models for Enzyme substrate complex
B-Enzyme complementary to Transition
Sate
 Proposed by Linus Pauling in 1946
an enzyme must be complementary to the
reaction transition state.
Models for Enzyme substrate complex
C-Induced Fit Model
 Proposed by Danial Koshland in 1958
 Substrate binding induce conformational
changes
Cofactors
Non-proteinous substances that associate with
enzymes. A cofactor is essential for the functioning
of an enzyme.
Cofactors
There are three kinds of cofactors present in
enzymes:
1-Prosthetic groups: These are cofactors tightly
bound to an enzyme at all times. A FAD is a
prosthetic group present in many enzymes.
1-Prosthetic groups:
Glucose Oxidase
Cofactors
2-Coenzyme: A coenzyme is bound to an enzyme only
during catalysis. At all other times, it is detached from the
enzyme. NAD+ is a common coenzyme.
Glucosidase converts maltose
into two molecules of glucose
Coenzymes
Are small Organic molecules -
derivatives of vitamins B complex
Examples: The thiamine (vitamin B1)-
dependent- Transketolase breakdown Glucose
by Pentose Phosphate Pathway.
Cofactors
3-Metal ions: For the catalysis of certain enzymes, a metal ion is required
at the active site to form coordinate bonds. Zn2+ is a metal ion cofactor
used by a number of enzymes.
Cofactors
Cofactors
Metals ions
 Fe, Co, Cu, Mg, Mn, and Zn
 Metalloenzymes
 Examples:
Catalase – Ferric ion
Glucose 6-Phosphatase –
Magnesium ion
Structure of Enzymes
Apoenzyme:
An inactive enzyme without its
cofactor
Inactive
Enzymes
Enzymes
Structure of Enzymes
Holoenzyme
An active enzyme together with its
cofactor
Inactive
Active
Properties of Enzymes
Three distinctive
features
1. Catalytic
Power
2. Specificity
3. Regulation
Enzymes
1. Catalytic Power
Enzymes
 Catalyze all biochemical reactions effectively
 Neither consumed nor permanently altered
 Enzyme-catalyzed reactions are highly efficient,
proceeding from 103–108 times faster than uncatalyzed
reactions.
The number of molecules of substrate converted to product
per enzyme molecule per second is called the
number, or kcat , and typically is 102–104 s-1.
turnover
2. Specificity
Enzymes are highly specific in their action when
compared with the chemical catalysts.
Substrate specificity : The substrate Specificity varies from
enzyme to enzyme.
a-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.
b-Relative substrate specificity :
 Some enzymes act on structurally related substances.
 This is dependent on the specific group or a bond present.
Trypsin hydrolyses peptide linkage involving Arginine or
Lysine.
Chymotrypsin cleaves peptide bonds attached to aromatic
amino acids (Phenylalanine, Tyrosine and Tryptophan)
 Glycosidases acting on glycosidic bonds of carbohydrates
 lipases cleaving ester bonds of lipids etc.
C-Broad specificity : Some enzymes act on
closely related substrates which is commonly
known as broad substrate specificity
e.g. hexokinase acts on glucose,
fructose/mannose and glucosamine but not on
galactose.
Enzymes
Regulation
Metabolic activators and
inhibitors
Classification of Enzymes
Enzymes
1Oxidoreductase
 catalyzes the transfer of electrons
 cofactors NAD/NADH2, FAD/FADH2
 Dehydrogenase, oxidases, peroxidases,
reductases
2Transferases
Alanine
transamina
se
 2 substrate
 Transferase,
Kinases
3Hydrolase
 Water act as nucleophile
 Phosphatase, nuclease, protease,
peptidase
4Lyase
Histidine
ammonia
lyase
 Cleave C-C, C-O, C-N, P-O by hydrolysis and oxidation
 This often forms a new double bond or ring structure
5Isomerases
 Re-arrangement without loss/add
 Racemase, isomerase,
phosphoglucomutase
5Isomerases
6Ligase
 catalyze the joining of two molecules
 ATP input, irreversible reaction
 Synthetase
6Ligase
Structure of Enzymes
Isozymes
 Variant of same enzyme
 Catalyze the same reaction
 Differ in amino acid sequence
 Often have different tissue
distribution
Structure of Enzymes
Enzymes
Lactate dehydrogenase -tetramer-two different subunits H and M
Structure of Enzymes
Isozymes:
HHMM
Enzymes
Creatine Phosphokinase ( CPK)
 Enzyme consist of two sub units.
 Catalyse inter-conversion of phosphocreatine into
creatine
Creatin
e
Phosphocreati
ne
Creatine Phosphokinase ( CPK)
Estimation of the CPK2 (MB) is the earliest reliable
indication of myocardial infarction.
In healthy individuals, the isoenzyme CPK2 (MB) is
almost undetectable in serum with less than 2% of total
CPK.
After the myocardial infarction( Ml ) , within the first 6-18
hours, CPK2 increases in the serum upto 20%
 CPK2 is not elevated in skeletal muscle disorders.
Clinical significance of Enzymes
What can measure?
1-Direct
measurement-Enzyme
quantity
2-Indirect
measurement-Enzyme
activity (Product quantity)
Clinical significance of Enzymes
Where can
measure? Different
Body Fluids:
Blood/Serum
Urine
Cerebrospinal
fluids
Tissues
Clinical significance of Enzymes
What is Unit of Measurement?
 Not express in mg or ug
 Express according to I.U system
One IU is the amount of enzyme to produce 1
umol of product per minute.
One Katal is the amount of enzyme for the
conversion of 1 mol of substrate per second
 1 IU= 16.67 X 10-9 Kat
Clinical significance of Enzymes
Why measure?
Normal Plasma levels reflects the balance
between
the rate of enzyme synthesis/release
into plasma during cell turnover
 the rate of clearance from the plasma
Clinical significance of Enzymes
Why measure?
Plasma level
increase
 Cell proliferation or Cell damage

Reduce clearance from circulation
(excretion by Kidney and abnormal
metabolism in Liver)
Clinical significance of Enzymes
Why measure?
Plasma level
decrease
 Reduce synthesis of enzyme
 Congenital deficiency
 Inherited variant of low
activity
Clinical significance of Enzymes
Measurement of enzymes is
helpful in
 Diagnosis
 Prognosis
 Localization of damages
Diagnostic importance of Enzymes
Estimation of enzyme activities in biological
fluids (particularly plasma/serum) is of great
clinical importance.
Enzymes in the circulation are divided into two
groups
 Plasma functional
 Plasma non-functional
1- Plasma specific or Plasma functional enzymes
 Normally present in the plasma
Enzyme activities are higher in plasma than in the
tissues.
They are mostly synthesized in the liver and enter the
circulation e.g. lipoprotein lipase, plasmin, thrombin,
choline esterase, ceruloplasmin etc.
lmpairment in liver function or genetic disorders often leads to
a fall in the activities of plasma functional enzymes e.g'
deficiency of ceruloplasmin in Wilson's disease
2- Non-Plasma specific or Plasma non-functional
enzymes
These enzymes are either totally absent or
present at a low concentration in plasma compared
to their levels found in the tissues.
Estimation of the activities of non-plasma specific
enzymes is very important for the diagnosis and
prognosis of several diseases.
Clinical significance of Enzymes
Therapeutic Enzymes
Summary
 Define what are Enzymes
 Describe Properties of Enzymes
 Classify Enzymes
 Discuss Structure of Enzymes
 Justify Clinical Significance of
Enzymes
Books
Chapters of Enzyme from
 Harper’s Biochemistry
 Lippincotts Illustrated Review:
Biochemistry