ENZYME BIOCHEMISTRY

1. ENZYME
S

2. CONTEN
TS Chemistry
 Nomenclature
 Classification
 Mechanism of EnzymeAction
 Enzyme Kinetics
 Factors affecting Enzyme Activity
 Activation
 Inhibition
 Regulation
 Diagnostic & Therapeutic uses of Enzymes

3. CHEMISTR
Y

4. Introduction
 Enzymes are biological catalysts that speed
up the rate of the biochemical reaction.
 Most enzymes are three dimensional globular
proteins (tertiary and quaternary structure).
 Some special RNA species
enzymes and are called Ribozymes
also act as
e.g.
hammerhead ribozyme.
Hammerhead enzyme

5. CHARACTERISTICS
 Enzymes speed up the reaction by lowering the activation
energy of the reaction.
 Enzymes only expedite the reaction & do not cause reactions
to take place.
 Their presence does not effect the nature and properties of
end product.
 They are highly specific in their action that is each enzyme
can catalyze one kind of substrate.
 Small amount of enzymes can accelerate chemical reactions.
 Enzymes are sensitive to change in pH, temperature and
substrate concentration.
 Turnover number is defined as the number of substrate
molecules transformed per minute by one enzyme molecule.
Catalase turnover number = 6 x106/min

6. 4
How do enzymes Work?
Enzymes work by
weakening
bonds which
lowers
activation
energy

7. 5
Enzymes
Free
Energy
Reactants
Products
Progress of the reaction
Free energy of activation
Without Enzyme
With Enzyme

8. SUBSTRATE
 The reactant in biochemical reaction is termed as substrate.
 When a substrate binds to an enzyme it forms an enzyme-
substrate complex.
EnzymeJoinsSubstrate

9. STRUCTURE OF ENZYMES
 The active site of an enzyme is the region that binds
substrates, co-factors and prosthetic groups and contains
residue that helps to hold the substrate.
 Active sites generally occupy less than 5% of the total surface
area of enzyme.
 Active site has a specific shape due to tertiary structure of
protein.
 A change in the shape of protein affects the shape of active
site and function of the enzyme.

10. 8
Active Site
Enzyme
Substrate
• An enzyme pocket/cleft formed by
folding of protein,contain a.a.side
chains,that participate in substrate
binding n catalysis
• Active Site

11. ACTIVE SITE
It chooses the substrate
and binds it to active site.
It performs the catalytic
action of enzyme.
o Active site can be further divided into:
Active Site
Binding Site Catalytic Site

12. LOCK AND KEY MODEL
 Proposed by EMIL FISCHER in 1894.
 Lock and key hypothesis assumes the active site of an
enzymes are rigid in its shape.
 There is no change in the active site before and after a
chemical reaction.

13. LOCK AND KEY MODEL
• In the lock- and- key model
- The active site has a rigidshape
- only substrates with the matching shape can fit
-The substrate is a keythat fits the lockof the active site
-This is an older model, however, and does not work for all enzymes

14. INDUCED FIT MODEL
 More recent studies have revealed that the process is much
more likely to involve an induced fit model (proposed by
DANIAL KOSH LAND in 1958).
 According to this exposure of an enzyme to substrate cause a
change in enzyme, which causes the active site to change it‟s
shape to allow enzyme and substrate to bind.

15. INDUCED FIT MODEL
• In the induced-fit model
-The active site is flexible, not rigid
-The shapes of the enzyme, active site, and substrate adjust to maximize the
fit, which improves catalysis
-There is a greater range of substrate specificity
This model is more consistent with a wider range of enzymes

16. 32
INDUCED FIT MODEL

17. CO-FACTORS
o Co-factor is the non protein molecule which carries out
chemical reactions that can not be performed by standard 20
amino acids.
o Co-factors are of two types:
 Organic co-factors
 Inorganic cofactors

18. H2 O + Co2
H2 CO3

19. INORGANIC CO-FACTORS
o These are the inorganic metal ions required for the proper
activity of enzymes.
Examples:
 Enzyme carbonic anhydrase requires Zn for its activity.
 Hexokinase has co-factor Mg
ORGANIC CO-FACTORS/CO-ENZY
o These are the organic molecules required for the proper
activity of enzymes.
Example:
 Glycogen phosphorylase requires the small organic
molecule pyridoxal phosphate.
++
++

20. CO-FACTORS
CO-FACTORS EXAMPLES:
Cu2+ (Cytochrome oxidase),
Fe2+,Fe3+ (Cytochrome oxidase,Catalase,Peroxidase),
K+(Pyruvate Kinase)
Mg2+(Hexokinase),Mn2+(Arginase),Ni2+(Urease),
Se(Glutathion peroxidase),Zn2+(Carbonic anhydrase).
CO-ENZYMES-EXAMPLES: (Vit.B DERIVATIVES)
NAD,NADP
FMN,FAD
TPP
PYRIDOXAL PHOS.
Co.A
Biotin
THF

23. PROSTHATIC GROUP
Prosthetic Group
o A prosthetic group is a cofactor or coenzyme that is
tightly/permanantly bound to the enzyme protein e.g.
Zn,Mg,Cu,Co,Mn,Se----------Co-factors
Vit.B derivatives,FAD-------------Co-enzymes
COSUBSTRATE
It is a cofactor or coenzyme that is looslly/transiently bound to an
enzyme e.g. NAD+ .

24. FAD+ : a prosthetic group of
succinate dehydrogenase
Binds
tightly
to the
enzyme

26.  Apoenzyme: is the protein part of the enzyme (its non protein
part- co-factor ,inorg.&org. removed).
.
o Holoenzyme: The complete complex of a protein with all
necessary small organic molecules, metal ions and other
components is termed as holoenzyme of holoprotein.
Continued…

27. SITES OF ENZYME SYNTHESIS
o Enzymes are synthesized by ribosomes which are attached to
the rough endoplasmic reticulum.
o Information for the synthesis of enzyme is carried by DNA.
o Amino acids are bonded together to form specific enzyme
according to the DNA‟s codes.

28. NOMENCLATURE

29. NOMENCLATURE
Each enzyme is assigned two names.
The first is its short, recommended name,convenient for everyday
use.
The second is the more complete systematic name,
which is used when an enzyme must be identified without ambiguity.

30. NOMENCLATURE –Recommended Name
• Most commonly used enzyme names have the
suffix–ase attached to the substrate of the reaction,
For example, sucrase catalyzes the hydrolysis of
sucrose,glucosidase,urease etc.
• Some names also describes the function of the
enzyme. For example, Lactate dehydrogenase
or Alcohol dehydrogenase .
• Some enzymes retain their original trivial names,
such as pepsin and trypsin.
l

31. EXAMPLES
substrate enzymes products
lactose lactase glucose + galactose
maltose maltase Glucose
cellulose cellulase Glucose
lipid lipase Glycerol + fatty acid
starch amylase Maltose
protein protease Peptides +
polypeptide

32. NOMENCLATURE-Systamatic Name
(IUB system of enzyme classification)
 A systematic classification of enzymes has been developed by IUB
(International Union of Biochemistry)-through
International Enzyme Commission.
o Enzymes are divided into six major classes each with numerous
subgroups.For a given enzyme ,the suffix –ase is attached to a
fairly complete description of the chemical reaction
catalyzed,including the names of all the substrates,for
example,Lactate NAD oxidoreductase.
o Each enzyme is also assigned a 4 digit EC no.1st digit repersent
class,2nd subclass,3rd subsub class and 4th individual enzyme,
o e.g. Lactate dehydrogenase is 1.1.1.27.
o The systamatic names are unambigous and informative but are
difficult for common use.

33. CLASSIFICATIO
N

34. CLASSIFICATION OF ENZYMES
 A systematic classification of enzymes has been developed by
IUB (International Union of Biochemistry)-through
International Enzyme Commission.
 This classification is based on the type of reactions catalyzed
by enzymes.
 There are six major classes.(OTHLIL)
 Each class is further divided into sub classes, sub sub-classes
and so on, to describe the huge number of different enzyme-
catalyzed reactions.

35. ENZYME CLASS REACTION TYPE EXAMPLES
Oxidoreductases Reduction-oxidation
(redox)
Lactate
dehydrogenase
Transferases Move chemical group Hexokinase
Hydrolases Hydrolysis; bond
cleavage with transfer
of functional group of
water
Lysosomal enzymes
Digestive enzymes
Lysases Non-hydrolytic bond
cleavage
Fumarase
Isomerases Intramolecular group
transfer
(isomerization)
Triose phosphate
isomerase
Ligases Synthesis of new
covalent bond
between substrates,
using ATPhydrolysis
RNA polymerase
Continued……..Classification of enzymes

36. Classification of enzymes

37. Classification of Enzymes

39. I. Oxidoreductases
These enzymes catalyse oxidation- reduction reactions
by transfer of electrons. This group is further sub divided
into 5 subgroups i.e.
1-Oxidases
2-Dehydrogenases
3-Hydroperoxidases
4-Oxygenases
5- Reductases

40. 1. Oxidases
In reactions catalyzed by these enzymes,
oxygen is added to H-atoms removed from
the substrate for e.g. ascorbic acid
oxidase.

41. O = C
I
HO – C
II
HO – C O
I
H - C -
I
HO – C – H
I
CH2OH
Dehydroascorbic acid
½ O 2 H2 o
Ascorbic acid ( vit C)
O = C
I
O – C O
I
O – C
I
H - C
I
HO – C – H
I
CH2OH
Action of Ascorbic acid oxidase

42. Xanthene oxidase
X.Oxidase X.Oxidase
Hypoxanthine----------------------Xanthine----------------Uric Acid
H2O+O2----H2O2 H2O+O2----H2O2

43. 2. Aerobic Dehydrogenase
These enzymes catalyze the removal of H2
from a substrate & can use either oxygen or
substances like methylene blue as H2
acceptors. e.g. Glucose oxidase which
catalyses the oxidation of glucose to
gluconolactone.

44. Action of Glucose Oxidase

45. 3. Anaerobic Dehydrogenases
These enzymes catalyse the removal of hydrogen
from the substrate & are unable to use oxygen as
hydrogen acceptor.
The H-acceptors take the H-atoms e.g. NAD+ ,
NADP+ and FAD all these three substances act as
co-enzymes for their respective enzymes.

46. GlycerAldehyde-3-Phosphate Dehydrogenase
During glycolysis:
Gly.alde.3-p Dehydrogenase
Glyceraldehyde-3-phos---------------------------1,3,Bisphosphoglycerate
NAD+---------------NADH2

47. COOH
I
C = O + NADH + H+
I
CH3
Pyruvic acid
Lactate dehydrogenase
COOH
I
HO - C - H + NAD+
I
CH3
Lactic acid

48. Succinate Dehydrogenase
Succinate Dehydrogenase
Succinic acid + FAD----------------------------------Fumaric acid +FADH2

49. 4. Hydroperoxidases
There are 2 enzymes in this class called
peroxidase + catalase. Catalyze the
decomposition of H2 O2.
Catalase Catalyze the following reaction:
2H2O2 2H2O + O2

50. 5. Oxygenases
 These enzymes catalyze the incorporation of
molecular O2 into the substrate. An example is
the conversion of phenylalanine to tyrosine by
the enzyme,
phenylalanine hydroxylase (monooxygenase).

51. H
I
CH2 - C – COOH
I I
NH2
H
I
CH2 - C – COOH
I I
NH2 + NADP+
I
OH
+ NADPH + H+ -----------→
O 2 H2 o
Phenylalamine
Tyrosine

52. Cytochrome P450 is also monooxygenase,
function for detoxification of many drugs.

53. 6. Reductase
These enzymes catalyze the reduction of their
substrate by adding H-atoms. e.g. glutathione
reductase which catalyze the conversion of
oxidised glutathione to reduced glutathione.

54. - Glu - Cys - Gly
I
S
I
S
I 2 - Glu - Cys – Gly + NADP+
- Glu - Cys - Gly I
SH
+NADPH+H+

55. II. Transferases
these bring about an exchange of functional
groups such as phosphates, amino, acyl &
methyl b/w 2 compounds.
Different types of transferases are:
1. Transaminases: These catalyze the exchange
Of –NH2 gp b/w an amino & a ketoacid. The
ketoacid becomes amino acid & the amino acid
becomes ketoacid.

56. e.g.
COOH COOH
І І
H2NCH + C = O
І І
CH2 CH2
І І
CH2 COOH
І
COOH
COOH COOH
І І
C = O + H2N C H
І І
CH2 CH2
І І
CH2 COOH
І
COOH
Glutamic acid Oxaloocetic acid Ketoglutamic acid Aspartic acid
Glutamic – Oxaloocetic
Transaninase
(Got/AST)

57. COOH COOH
І І
C = O + H2N C H
І І
CH2 CH3
І І
CH2
І
COOH
Pyruic acid Ketoglutaric acid Alanine
Glutamic Pyruic
Transaninase
(GPT/ALT)
e.g.
COOH COOH
І І
H2NCH + C = O
І І
CH2 CH3
І
CH2
І
COOH
Glutamic acid

58. 2. Phosphotransferases(kinases):These catalyse
the transfer of PO4 gps. & are called kinases.
e.g. hexokinase which catalyze the reaction,
Glucose + ATP → glucose 6 PO4+ ADP

59. 3. Transmethylases: These catalyze the transfer of
methyl gps. e.g. conversion of noradrenaline to
adrenaline

60. CHOH – CH2 – N - H
I I
H CH3
OH -
I
OH
CHOH – CH2 – N - H
I I
CH3
OH -
I
OH
Noradrenaline
Adrenaline

61. 4. Transpeptidases: These catalyze the transfer of
aminoacids or peptides e.g. formation of
hippuric acid from benzyl CoA + glycine

62. O = C – S – CoA
I
+ H2N – CH2 – COOH
glycine
Benzyl CoA
O = C – N - COOH
I I
H
+ CoA - SH
Benzyl glycine or Hippuric acid

63. 5. Transacylases: These catalyze the transfer of
acyl gps. e.g Choline acetyltransferase which
catalyzes the synthesis of acetylcholine
Acetyl – CoA + Choline → acetylcholine + CoA

64. III. Hydrolases
 Catalyze hydrolosis i.e add H2o molecule to the
substrate + Simultaneously decompose it.
Various sub-groups are:
1. Protein hydrolyzing enzymes i.e proteinases or
proteases or proteolytic enzymes
A. Expopeptidases: Catalyze the hydrolysis of
terminal peptide bonds
B. Endopepridases: Attack the centrally located
peptide bonds

65. 2. Carbohydrases
catalyze the hydrolysis of the glycosidic bonds
e.g. enzyme amylase converts starch to
maltose. Maltose is further hydrolysed by the
enzyme maltase to glucose. Sucrase, lactase &
cellulase also are examples of this gp. of
enzyme.

66. 3. Lipid hydrolyzing enzymes: e.g.
i. Lipases: Act on triglycerides or neutral fats to
liberate glycerol, F.A + monoglycerides +
diglycerides. Pancreatic lipase is very impt. In
the digestion of fats.
ii. Cholesteryl esterase: Hydrolyses cholesterol
esters.
iii. Phospholipase: Also known as lecithinases +
splits lecithins as well as cephalins

67. iv.Lyases:
Catalyze the addition of NH3, H2O or CO2 to
double bonds or the removal of these from double
bonds e.g. conversion of fumaric acid to L- malic
acid

68. LYASES
These enzyme catalyse the addition of CO2,H2O OR NH3 to double
bonds or removal of these gps from double bonds.e.g.
Fumarase
Fumaric acid + H2O------------L.Malic acid

69. COOH COOH
I fumerase I
CH + H2O HOCH
I I I
HC CH2
I I
COOH Fumaric acid COOH
L- Malic acid

70. v. Isomerases:
vi. Catalyze the transfer of gps. With in molecules to yield isomeric
forms of the substrate . eg.
Glucose 6 - PO4 fructose 6- PO4
phosphohexose isomerase

71. ISOMERASE
Trios-p-Isomerase
GlycerAldehyde-3-phosphate<----------DiHydroxyAcetone-Phosphate

72. vi.Ligases:
Catalys synthetic reactions,condensation,usually
ATP dependent, joining 2 mol by forming C- O, C –
S, C - N & C - C bond

73. e.g.
CH3 COOH
I I
C = O + CO2 + ATP CH2 + ADP +P1
I I
SCOA C = O
I
S - CoA
Acetyl CoA Malonyl coA
Acetyl – CoA carboxylase

74. Succinate thiokinase
Succininic acid+ CoA-SH+GTP-----------------------Succinyl CoA