Enzymes are biological catalysts that speed up chemical reactions by lowering their activation energy. They are typically proteins but can also be RNA. Enzymes work by specifically binding substrates and facilitating their conversion to products. They are affected by factors like temperature, pH, substrate/product concentration, and can be inhibited in a reversible or irreversible manner. The three main types of inhibition are competitive, non-competitive, and uncompetitive inhibition which act by binding to the active site or altering the enzyme's structure.

1. Enzymes

2. Enzymes – coined by Frederick
“In Yeast”
• Biological catalyst
• Role of Catalyst : To speed up the reaction – Conversion of
substrate to products with out alter equilibrium
• Substrate - Molecules which are acted on.
• Products - Molecules which are produced.
• Eg: 4H+ O2 → 2H2o

3. Characteristics of Enzymes
All enzymes are proteins, Except Ribozyme- RNA act as catalyst (Non protein
enzyme)
Physical and chemical reaction –similar to protein
Heat labile
Water soluble
Precipitation by metals as similar to proteins
16% weight by nitrogen content
Enzymes can be of two types
 Simple Enzyme: Consists of only proteins.
 A complex enzyme consists of:
 Protein part: Apoenzyme
 Nonprotein part
 Apoenzyme + Nonprotein part (Prosthetic group/Cofactor/Coenzyme) = Holoenzyme

4. Differences
Enzyme
Complex proteins
High molecular weight
Catalyze specific reactions
Affected by temperature and pH
Regulated by specific substances
Can be easily inactivated
Catalyst
Simple inorganic molecules
Low molecular weight
Have wide range
No effect of temperature and pH
No regulation
Can’t be inactivated

5. Enzyme definitions Term Definition
Enzyme
(simple)
Protein only enzyme that facilitates a chemical reaction
Coenzyme Compound derived from a vitamin (e.g. NAD+) that assists an
enzyme in facilitating a chemical reaction
Cofactor Metal ion (e.g. Mg2+) that that assists an enzyme in facilitating a
chemical reaction
Apoenzyme Protein only part of an enzyme (e.g. isocitrate dehydrogenase)
that requires an additional coenzyme to facilitate a chemical
reaction (not functional alone)
Holoenzyme Combination of the apoenzyme and coenzyme which together
facilitating a chemical reaction (functional)

6. • Enzymes are named - type of the reaction they catalyze and/or their substrate(the reactant upon
which an specific enzyme acts )
• Suffix of an enzyme --ase
• lactase, amylase,lipase ,etc.,
• some digestive enzymes with suffix in - trypsin, chymotrypsin,etc
• Prefix--denotes the type of the reaction catalyzes
• oxidases, Hydrolases
• substrate identity is often used together with the reaction type.
• lactate dehydrogenase, pyruvate decarboxylase
Enzyme nomenclature

7. • The word monomeric enzyme is used if it is made up of a single
polypeptide e.g. ribonuclease, trypsin.
• Some of the enzymes which possess more than one polypeptide
(subunit) chain are known as oligomeric enzymes e.g. lactate
dehydrogenase, aspartate transcarbamoylase etc.
• There are certain multienzyme complexes possessing specific sites to
catalyse different reactions in a sequence.
• Only the native intact multienzyme complex is functionally active and
not the individual units, if they are separated
• e.g. pyruvate dehydrogenase, fatty acid synthase, prostaglandin
synthase

8. Principle of the international classification
• Each enzyme has classification number consisting of four
digits:
• Example, EC: (2.7.1.1) HEXOKINASE

9. 2 Class (Transferase)
7 Subclass (Transfer of Phosphate)
1 Sub-sub Class (Phosphotransferase with a
Hydroxyl group as Acceptor)
1 D-glucose as Phosphoryl group Acceptor
EC: (2.7.1.1)
ATP,D-HEXOSE-6-PHOSPHOTRANSFERASE (Hexokinase)

10. Classification of Enzymes
• According to the IUB system, enzymes are
classified into six major classes as follows:
1. EC-1 : Oxidoreductases
2. EC-2 : Transferases
3. EC-3 : Hydrolases
4. EC-4 : Lyases
5. EC-5 : Isomerases
6. EC-6 : Ligases.

11. EC-1 .Oxidoreductase
 Catalysing oxidation reduction reaction where one substrate
is oxidized and other is reduced (Transfer of hydrogen)
Subclassified into
 Dehydrogenase
 Oxygenase
 Monooxygenase
 Dioxygenase
 Oxidase
 Catalase
 Peroxidase.

12. Dehydrogenase
•Catalyze removal of hydrogen from the substrate
•Do not use oxygen as a hydrogen acceptor.
Example:
Alcohol Dehydrogenase
Lactate Dehydrogenase
Succinate Dehydrogenase.

13. Oxygenase
Monooxygenase
 Incorporate one atom of molecular
oxygen into the substrate.
Examples
 Phenylalanine Hydroxylases
 Tyrosine Hydroxylase
 Tryptophan Hydroxylase
 7 alpha Hydroxylases
 Cytochrome p450.
 Incorporate both atoms of
molecular Oxygen into the
substrate.
Examples
 Homogentisate oxidase
 Tryptophan Pyrrolase
(Dioxygenase)
 Nitric Oxide Synthase
Catalyse the direct transfer and incorporation of oxygen into a substrate
molecule
Dioxygenase

14. Oxidases
• Removal of hydrogen from a substrate with oxygen as the
acceptor of Hydrogen.
Examples are:
• Mono Amino Oxidase
• Cytochrome C Oxidase
• Xanthine Oxidase.

15. EC-2.Transferase
• Catalyze the transfer of a group such as, amino, carboxyl, methyl or phosphoryl,
etc. from one molecule to another are called transferases.
• Amino transferase or transaminase
• Kinase
• Transcarboxylase.
• Specific Example

16. EC-3 Hydrolases
• Enzymes of this class catalyze the cleavage of C-O, C-N, C-C and some
other bonds with the addition of water.
• Acid phosphatase
• All digestive enzymes like α-amylase, pepsin, trypsin, chymotrypsin, etc.

17. EC-4.Lyases
• Cleavage of C-O, C-C and C-N bonds by means other than hydrolysis or
oxidation, giving rise to compound with double bonds or catalyze the reverse
reaction, by the addition of group to a double bond.
• In cases where reverse reaction is important, then synthase, is used in the
name.

18. EC-5 Isomerase
• Isomerases catalyze intramolecular structural rearrangement in a
molecule.
• They are called epimerases, isomerases or mutases, depending on
the type of isomerism involved.
Examples : Phosphohexose isomerase

19. EC-6 Ligase
• Ligases catalyze the joining of two molecules coupled with the hydrolysis of ATP.
Example,
• Acetyl CoA carboxylase
• Arginosuccinate synthetase
• PRPP synthetase
• Carbamoyl phosphate Synthetase
• Glutamine synthetase

20. Mechanism of action of enzymes
 Active centre
 Specific Binding of substrate is explained by
different theories:
Michaelis–Menten theory
Fischer’s Template theory
Koshland’s Induced Fit theory

21. Michaelis–Menten Theory
(Enzyme–Substrate Complex Theory)
• Enzyme combines with a substrate to form a transient Enzyme-Substrate
Complex which immediately break into Enzyme and products.

22. Lock and Key Model or Rigid Template Model
• Proposed by Emil Fisher in 1890 called Fischer’s
Template Theory
• Three dimensional structure of the active site of
unbound enzyme is complementary to the substrate.
• The substrate fits into the active site in much the
same way that a key fits into a lock.
• Failed to explain dynamic changes that accompany
catalysis.

23. Koshland’s Induced Fit Theory
• Binding of substrate to specific part of
enzyme induce conformational changes in
the active site of the enzyme
• Enzyme changes shape during or after
binding with the substrate
• Can explain the dynamic changes that
accompany catalysis.

24. FACTORS AFFECTING ENZYME ACTIVITY
• 1. Concentration of enzyme---As the concentration of the enzyme is increased, the velocity of
the reaction proportionately increases.
• This property of enzyme is made use in determining the serum enzymes for the diagnosis of
diseases.
• By using a known volume of serum, and keeping all the other factors (substrate, pH,
temperature etc.) at the optimum level, the enzyme could be assayed in the laboratory

25. Enzyme concentration
• Velocity of enzyme reaction is directly
proportional to the enzyme concentration
• Enzyme activity is expressed as micromoles of
substrates converted to product per minute
under specific conditions
• Specific activity of an enzyme- No. of units of
enzymes per mg of protein
• Catalytic activity – turnover number – Units
of activity per mole of enzyme

26. 2. Concentration of substrate
• Increase in the substrate concentration gradually increases the velocity of enzyme reaction within
the limited range of substrate levels.
• A rectangular hyperbola is obtained when velocity is plotted against the substrate concentration.
• Order of reaction : When the velocity of the reaction is almost proportional to the substrate
concentration (i.e. [S] is less than Km), the rate of the reaction is said to be first order with respect
to substrate.
• When the [S] is much greater than Km, the rate of reaction is independent of substrate
concentration, and the reaction is said to be zero order.

27. Michaelis-Menten constant, Km
• Measure of the substrate concentration required for significant
catalysis to occur.
• Km is independent of enzyme concentration
• It is a measure of the affinity of the enzyme for its substrate
• Eg. Hexokinase and glucokinase
• A high Km indicates weak binding and a low Km indicates strong
binding with its substrate.

28. 3.Effect of temperature
• Velocity of an enzyme reaction increases with increase in
temperature up to a maximum and then declines.
• A bell-shaped curve is usually observed.
• Temperature coefficient or Q10 is defined as increase in enzyme
velocity when the temperature is increased by 10°C.

29. 4. Effect of pH
• Increase in the hydrogen ion concentration (pH) considerably influences the enzyme activity and a
bell-shaped curve is normally obtained.
• 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 enzyme
becomes totally inactive.
Example,Pepsin = 1.2 ,Trypsin = 8.0

30. 5. Effect of product concentration
• The accumulation of reaction products generally decreases the enzyme velocity.
• For certain enzymes, the products combine with the active site of enzyme and form a loose
• complex and, thus, inhibit the enzyme activity.
• In the living system, this type of inhibition is generally prevented by a quick removal of
• products formed.
• The end product inhibition by feedback mechanism.
• 6. Effect of activators
• Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+,
• Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc. for their optimum activity.
• Metals function as activators of enzyme velocity through various mechanisms—combining with
the substrate, formation of ES-metal complex, direct participation in the reaction and bringing a
conformational change in the enzyme.

31. • Two categories of enzymes requiring metals for their activity are
distinguished
• Metal-activated enzymes : The metal is not tightly held by the enzyme and can be
exchanged easily with other ions e.g. ATPase (Mg2+ and Ca2+) Enolase (Mg2+)
• Metalloenzymes : These enzymes hold the metals rather tightly which are not readily
exchanged. e.g. alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase,
carboxypeptidase and aldolase contain zinc.
• Phenol oxidase (copper);
• Pyruvate oxidase (manganese);
• Xanthine oxidase (molybdenum);
• Cytochrome oxidase (iron and copper).
• 7. Effect of time Under ideal and optimal conditions (like pH, temperature etc.), the
time required for an enzyme reaction is less. Variations in the time of the reaction are
generally related to the alterations in pH and temperature.

32. • 8. Effect of light and radiation
• Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes due to
the formation of peroxides. e.g. UV rays inhibit salivary amylase activity.
• 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.
• The existence of active site is due to the tertiary structure of protein resulting in three
dimensional native conformation.
• Active sites are regarded as clefts or crevices or pockets occupying a small region in a big
enzyme molecule.
• It is flexible,the active site possesses a substrate binding site and a catalytic site.
• The coenzymes or cofactors on which some enzymes depend are present as a part of
the catalytic site.
• 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.
• E + S------->ES------> E + P

35. Enzyme Inhibition
• Enzyme inhibitor is defined as a substance which binds with the
enzyme and brings about a decrease in catalytic activity of that
enzyme.
• The inhibitor may be organic or inorganic in nature.
• There are three broad categories of enzyme inhibition
• 1. Reversible inhibition.
• 2. Irreversible inhibition.
• 3. Allosteric inhibition.

38. • A competitive inhibitor (CI)resembles the substrate – Inhibitor competes with
the substrate for binding to
• the active site of the enzyme
• – If an inhibitor is bound to the active site: Prevents the substrate molecules to
access the active site
• – Decreasing / stopping enzyme activity
• • The binding of the competitive inhibitor to the active site is a reversible process
– Add much more substrate to outcompete the competitive inhibitor
• Many drugs are competitive inhibitors:
• – Anti-histamines inhibit histidine decarboxylase, which converts histidine to
histamine
• Succinate dehydrogenase--succinate substrate ---analogue--malonic acid(CI)
• In competitive inhibition,

39. Competitive inhibitor

41. • A non-competitive inhibitor decreases enzyme activity by binding to a site on the enzyme
other than the active site
• – The non-competitive inhibitor alters the tertiary structure of the enzyme & the active site
• Decreasing enzyme activity
• Substrate cannot fit into active site– Process can be reversed only by lowering the
non-competitive inhibitor.
• • Example: – Heavy metals Pb2+ & Hg2+ bind to –SH of Cysteine, away from active site
• Disrupt the secondary & tertiary structure
• For non-competitive inhibition,
Non Competitive Inhibitor

42. Non Competitive Inhibitor

44. iii.UnCompetitive inhibition

 Not to substrate
 Eg. Alkaline Phosphatase
Suicide Inhibition
 Special type of irrreversible inhibition
 Mechanism based inactivation
 Some product in the pathway – inhibit
 Eg. Ornithine decarboxylase - Ornithin to putrescine;
 But Difluro methyl ornithine inhibits ODC in
Trypanasomiasis
 5 fluro uracil inhibits DNA synthesis
 Allopurinol inhibits xanthine oxidase
 Aspirin - Cyclooxygenase

45. IRREVERSIBLE INHIBITOR
An irreversible inhibitor binds with an enzyme
tightly covalently and forms a stable complex.
An irreversible inhibitor cannot be released by
dilution or simply by increasing the
concentration of substrate.

46. Irreversible inhibitors can be divided into three
categories:
I.Substrate analogue inhibitor or affinity labels
II.Group specific inhibitors
III.Suicide inhibitor or mechanism based inactivation.

47. In terms of enzyme kinetics, the effect of an
irreversible inhibitor is like that of the reversible non-
competitive inhibitors resulting in a decreased in Vmax but
having no effect on the Km.

48. I. Substrate Analogue Irreversible Inhibitor
( or ) Affinity Labels
Substrate analogues or affinity labels are molecules
that are structurally similar to the substrate.
These substrate analogues possess a highly reactive group
which is not present in the natural substrate.
 The reactive group of substrate analogues covalently reacts with
amino acid residues of the active site of the enzyme and
permanently block the active site of the enzyme
 3-Bromoacetal phosphate (BAP) inhibits enzyme phosphotriose
isomerase of glycolysis.

49. II. Group Specific Irreversible
Inhibitor
 These inhibitors react with specific R-groups
(side chain) of amino acid residues in the active site of enzyme.
 Examples of group specific irreversible inhibitors:
– Di-isopropylphosphofluoride (DIPF)
– Iodoacetamide
– Heavy metals

50. a) Di-isopropylphosphofluoride (DIPF)
 DIPF can inhibit an enzyme acetylcholine esterase by covalently reacting with
hydroxyl group of a serine residue present at the active site of the enzyme
 DIPF has also been found to inhibit trypsin, chymotrypsin, elastase and
phosphoglucomutase.
 Irreversible inhibition of acetylcholinesterase by a group-specific inhibitor,
diisopropylphosphofluoride (DIPF).

51.  Iodoacetamide and heavy metals like, Pb2+, Ag+,
Hg2+, etc. which react with sulfhydryl (-SH) group of cysteine
residues present at the active site of the enzyme and makes
them inactive.

52. III.Suicide Inhibitor or Mechanism Based inactivation
 These compounds are relatively unreactive until they bind to
the active site of a specific enzyme.
 On binding to the active site of the enzyme they carry out the
first few catalytic activities of the normal enzyme reaction.
 Instead of being transformed into a normal product, however,
the inhibitor is converted to a very reactive compound that
combines irreversibly with the enzyme leading to its
irreversible inhibition
 These are also called mechanism based inactivation because
they utilize the normal enzyme reaction mechanism to
inactivate the enzyme.

53. Example Suicide Inhibitor
Penicillin
Inactivates bacterial enzyme glycopeptidyl transpeptidase
involved in the formation of bacterial cell wall.
Aspirin
Inactivates an enzyme cyclo-oxygenase required for the
synthesis of prostaglandins .

54. Clinical Application of EnzymeInhibitor
 Enzyme inhibitors have therapeutic applications.
 Most antibiotics and anticancer drugs that are used
therapeutically are either competitive inhibitor or mechanism
based suicide inhibitor.

56. ALLOSTERIC ENZYME
Allosteric enzyme is a regulatory enzyme.
The term allosteric derives from Greek word, allo means
other and steros means space or site.
Allosteric enzymes are those having other site in addition to
active site for binding of modulator (regulatory metabolites).
Allosteric enzymes may be inhibited or stimulated by their
modulators
Modulators that inhibit enzyme activity are termed
negative modulators. Whereas those that increase enzyme
activity are called positive modulators.

58. Feed back control
• A process in which activation or inhibition of one of the earlier
reaction
• steps in a reaction sequence is controlled by a product of this reaction
sequence.
– One of the mechanisms in which allosteric enzymes are regulated
– Most biochemical processes proceed in several steps & each step is
catalyzed by a different enzyme
• The product of each step is the substrate for the next step / enzyme.

59. Feed back control
Example:
The degradation of glucose through a metabolic pathway can be regulated in several ways,
1.The enzyme PFK is allosterically inhibited by the product ATP.
2.Glycolysis (makes ATP) is slowed when cellular ATP is in excess.

60. Enzyme Specificity
• Absolute Specificity
– An enzyme will catalyze a particular reaction for only one substrate
– Most restrictive of all specificities
• Not common
– Catalase has absolute specificity for hydrogen peroxide (H2O2)
– Urease catalyzes only the hydrolysis of urea
• Group Specificity
– The enzyme will act only on similar substrates that have a specific functional group
• Carboxypeptidase cleaves amino acids one at a time from the carboxyl end of the
peptide chain
• Hexokinase adds a phosphate group to hexoses

61. Enzyme Specificity
• Linkage Specificity
– The enzyme will act on a particular type of chemical bond, irrespective of the rest of the
molecular structure
– The most general of the enzyme specificities
• Phosphatases hydrolyze phosphate–ester bonds in all types of phosphate esters
• Chymotrypsin catalyzes the hydrolysis of peptide bonds
• Stereochemical Specificity
– The enzyme can distinguish between stereoisomers
– Chirality is inherent in an active site (as amino acids are chiral compounds)
• L-Amino-acid oxidase catalyzes reactions of L-amino acids but not D-amino acids

62. Proteolytic Enzymes & Zymogens
• 2nd mechanism of allosteric enzyme regulation
– Production of an enzyme in an inactive form
– Activated when required (right time & place)
• Proteolytic enzymes catalyze breaking of peptide bond in proteins
– To prevent these enzymes from destroying the tissues, that produced them, they are released in
an inactive form = ZYMOGENS

64. • Most digestive & blood-clotting enzymes are proteolytic
– Blood clotting enzymes break down proteins within the blood so that they can form the clot
• Platelets interspersed with tangled protein (collagen and thrombin)
• Activation of a zymogen requires the removal of a peptide fragment from the zymogen structure
– Changing the 3-D shape & affecting the active site
• E.g. Pepsiongen (zymogen)--------->Pepsin (active proteolytic enzyme)

65. Covalent Modification of Enzymes
• Covalent modifications are the 3rd mechanism of enzyme activity regulation
– A process of altering enzyme activity by covalently modifying the structure of the enzyme
by Adding / removing a group to / from the enzyme
• Most common covalent modification = addition & removal of phosphate group:
– Phosphate group is often derived from an ATP molecule
• Addition of phosphate = phosphorylation is catalyzed by a Kinase enzyme
• Removal of phosphate = dephosphorylation is catalyzed by a Phosphatase enzyme
– Glycogen synthase: involved in synthesis of glycogen
• Deactivated by phosphorylation
– Glycogen phosphorylase: involved in breakdown of glycogen
• Activated by phosphorylation

66. coenzymes
 The non protein, organic, low molecular weight and dialysable substance associated with
enzyme function is known as coenzyme
 A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein is
called a prosthetic group.
 A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions is
called a holoenzyme.
 The protein part of such an enzyme is called the apoenzyme or apoprotein.
 Low molecular weight organic substances
 Act as transient carriers of specific functional groups.
 Most are derived from vitamins B complex, organic nutrients required in small amounts in the
diet.

68. Cofactors

69. Isoenzymes
• Isoenzyme catalyze the same reaction in different tissues in the body
– e.g. lactate dehydrogenase (LDH) consists of 5 isoenzymes, other example CREATINE KINASE, ALP
• Each isoenzyme of LDH has the same function
– Converts lactate to pyruvate
• LDH1 isoenzyme is more prevalent in heart muscle
• LDH5 form is found in skeletal muscle & liver
• Isoenzymes can be used to identify the damaged or diseased organ or tissue
• It is a marker for a particular location
• If LDH1 isoenzyme was found in the blood >>> indicates heat muscle damage

70. Isoenzymes-LDH

72. Isoenzymes (Isozymes)
 Physically distinct and separable forms of the enzyme with same catalytic activity
 Formed by defect in structural genes.
 Features
1. multiple molecular forms of an enzyme
2. products of closely related genes
3. may be originated from different tissues
eg: CK-1 = brain
CK-2 = heart
CK-3 = skeletal muscle
4. study of isoenzymes is useful to understand the disease of different organs

73. Types of Isozymes
 True isozymes
 Hybrid isozymes
 Allozymes
 Isoforms
 1. True iso-enzymes
 Iso-enzymes are products of different genes
 Genes may be located on same chromosome (eg: cytoplasmic
and mitochondrial malate dehydrogenase) or on different
chromosomes
(eg: salivary and pancreatic amylase)

74. Hybrid iso-enzymes
Isoenzymes arises from the enzymes having different subunits
Eg: Isoenzymes Subunits Tissue
LDH-1 H4 heart
LDH-2 H3M1 RBC
LDH-3 H2M2 brain
LDH-4 H1M3 liver
LDH-5 M4 skeletal
muscle

75. Allozymes
 Different forms of enzymes produced by same locus of the
gene
Eg: 400 forms of G-6-P-D are produced by same locus on the
X-Chromosome

76. Isoforms
 Arises from post-translational modification of enzymes
Eg: Isoforms of CK-MB
CK-MB1, CK-MB2
Sialic acid content in ALP

77. Identification of iso-enzymes
• Electrophoresis
LDH-1 = fastest mobility
LDH-5 = slowest mobility
• Heat stability
LDH-1 = not denatured at 600C
LDH-5 = denatured at 600C

84. UNITS OF ENZYME ACTIVITY
• KATAL- one kat denotes the conversion of one mole substrate per
second(mol/sec).Activity may also expressed as millikatals(mkat),
microkatals(ukat)
• INTERNATIONAL UNIT- one SI unit or international unit is defined as
the amount of enzyme activity that catalyses the conversion of one
micromol of substrate per minute. SI unit and Katal are
interconvertible.
• 1 IU = 60 uKatal
• or
• 1 mkatal = 1.67 IU