ENZYMES

OBJECTIVES
At the end of the lecture, the student should be
able to:
1. Define the following:
a. Enzymes
b. Apoenzyme
c. Coenzyme
d. Holoenzyme
e. Metalloenzyme
f. Regulatory enzyme
g. Active site of the enzyme
h. Allosteric site of the enzyme
i. Substrate
2. Discuss the helpers (cofactors) of enzymes.

OBJECTIVES
3. Enumerate the six major classes of enzymes.
4. Discuss the characteristics of enzymes.
5. Explain the models of enzyme-substrate
complex.
6. Explain enzyme kinetics.
a. Factors that affect enzyme activity or
reaction velocity.
b. Ways of expressing enzyme activity.
7. Discuss the operation and plots used to
illustrate enzyme kinetics.
a. Michaelis-Menten kinetics
b. Lineweaver-Burke Double Reciprocal Plot
c. Michaelis constant and its significance

OBJECTIVES
8. Discuss enzyme inhibition and its effect on
reaction velocity.
a. Reversible
b. Irreversible
9. Discuss the different ways of regulating enzyme
activity.
10. Explain the factors affecting enzyme activity.
11. Elucidate uses and clinical application of
enzymes.

ENZYMES
Specialized protein catalysts
that accelerate chemical
reactions

DEFINITION OF TERMS
APOENZYME

APOENZYME

APOENZYME

Coenzyme

Prosthetic
group

Metal
ion

HOLOENZYME

Protein
part

Cofactor
(Nonprotei
part)

ENZYME COFACTORS
A. Coenzyme

Enzyme

Chemical
Groups
Transferred

Vitamin
Precursor

Aldehydes

Thiamine
(Vit B1)

Thiamine
Pyrophosphate
(TPP)

Pyruvate dehydrogenase,
Isocitrate dehydrogenase, ketoglutarate dehydrogenase,
Transketolase, -Ketoacid
dehydrogenase

Flavin Adenine
Dinucleotide
(FAD)

Succinate dehydrogenase, Ketoglutarate dehydrogenase,

Pyruvate dehydrogenase,
Nitric oxide synthase

Electrons

Riboflavin
(Vit B2)

Nicotinamide
Adenine
Dinucleotide (NAD)

Lactate dehydrogenase;
Other dehydrogenases

Hydride ion
(:H-)

Nicotinic acid
(Niacin; B3)

Amino groups

Pyridoxine
(Vit B6)

Electrons and
acyl groups

Not required
in diet

Pyridoxal
Phosphate (PLP)
Lipoate

Glycogen phosphorylase,
-ALA synthase, Histidine
decardoxylase, Alanine
aminotransferase
Pyruvate dehydrogenase
-Ketoglutarate dehydrogenase

ENZYME COFACTORS
A. Coenzyme

Enzyme

Coenzyme A
(CoASH)

Acetyl CoA
carboxylase

Biocytin

Pyruvate
carboxylase,
Acetyl CoA
carboxylase,
Propionyl CoA
carboxylase

Chemical
Groups
Transferred
Acyl groups

Vitamin
Precursor
Pantothenic
acid & other
compounds

CO2
Biotin

5deoxycobalamin

Methylmalonyl
mutase

H atoms and
alkyl groups

Vit B12

One-carbon
groups

Folic acid

Tetrahydrofolalate

Thmidylate
synthase

CLASSES OF COENZYMES
CLASS

EXAMPLES

Activation-Transfer
Coenzymes

TPP
Coenzyme A
Biotin
PLP

Oxidation-Reduction
Coenzymes

NAD+
FAD
Vit E, Vit C
Marks Medical Biochemistry, 3rd ed.

ENZYME COFACTORS: COENZYME A

Pantothenic acid

1. Pantothenic acid-derived,
co-factor of several
enzymes like acetyl CoA
carboxylase.
2. Takes part in reactions of
the CAC, FA synthesis and
oxidation, acylations and
cholesterol synthesis.

CH3
O
H
I
II
C-CH2-CH2-N-C-CC-CH2O
I
II I I
NH
O OH CH3
O = P OI
I
CH2
O
O
I
II
NH2
I
CH2
N
N
O = P OH
I
I
SH
N
N
H
O
O
HH

Active
sulfhydryl
group that form
thioesters with
acyl groups

OH

O
I
O = P OI
O-

ENZYME COFACTORS:
COENZYME A
-Ketoglutarate Dehydrogenase
Complex
Coenzymes:
COOH1. TPP
2. Lipoic Acid
|
3. FAD
CH2 4. NAD+
5. CoASH
|

CH2
|
C=O
|
CoASH
COO-

-Ketoglutarate (C5)

Pyruvate decarboxylase
Dihydrolipoyl transacetylase
Dihydrolipoyl dehydrogenase

NAD

NADH
+ H+

CO2

G0 = - 8.0 kcal

COO|
CH2
|
CH2
|
C=O
|
S ~ CoA

Succinyl CoA (C4)

ENZYME COFACTORS:
COENZYME A
Pyruvate Dehydrogenase Complex
COO|
C=O
|
CH3

Pyruvate decarboxylase
TPP
Lipoate Dihydrolipoyl transacetylase
Dihydrolipoyl dehydrogenase
FAD

Pyruvate CoASH
NADH+ +
+
NAD
(C3)
H+

CO2

G0 = - 8.0 kcal/mole

S ~ CoA
|
C=O
|
CH3
Acetyl CoA
(C2)

Pyruvate

ENZYME COFACTORS: NAD & NADP

Lactate

I
H-O-C-H
I
Functional
3 CH3
2

group

O
II
C NH2

Nicotinamide
ring

H
C
Lactate
dehydrogenase

N1
O CH2 O
P
H H
H
H
O
O
OH OH

O
P

2
5

O CH2 O
4
1
H H
H3 2 H
3
2
OH
OH

Ribose
ring

O
II
C NH2

N
I
R

NH2
5
4

1N

3 2

Adenine
ring

NADP+ contains a P
on this 2-hydroxyl

AMP

Nicotinamide adenine dinucleotide (NAD+)

Keto
group

1 COO-

Dissociates
as H+

COOI
C=O + H+
I
CH3

AMP provides additional

binding interactions that
induce conformational
changes in the enzyme

ENZYME COFACTOR: NAD+

COO
|
HO - C H
|
CH3

L-Lactate

Lactate
dehydrogenase

NAD+

NADH+ + H+

COOI
C=O
I
CH3
Pyruvate

COENZYME:
BIOTIN
O
II

H-N
H

N-H

IIIIIIIIIIIIIII

IIIIIIIIIIIIIII

I
III
III
III
II

Biotin

R
III

H
Protein portion of enzyme:
Acetyl CoA carboxylase
Propionyl CoA carboxylase
Pyruvate carboxylase
N
H
O
O
IIII
H-N

NH
=O

N-H
IIIIIIIIII

Lysyl
residue

Biotin
III
III

CO2
H IIIIIIIIIIIIIII
attachment
site

C
II
O

III
II

1. One of the B complex

vitamins.
2. A cofactor of such
enzymes as acetyl
CoA carboxylase,
propionyl CoA
carboxylase and
pyruvate carboxylase.
3. It is a carrier of activated
CO2, hence involved in
carboxylation reactions.

COENZYME: BIOTIN
BIOTIN

COOI
C =O
I
CH3

Pyruvate

COO|
C= O
|
CH2
|
COO-

CO2
ATP

ADP + Pi

Pyruvate
carboxylase

Oxaloacetate

Gluconeogenesis

COENZYME:THIAMINE PYROPHOSPHATE
Reactive
carbon atom:
carrier of aldehyde
groups
H

Dissociable
proton

H3C

NN S
NH2
N
C
C
N
N

H
CH2

Coenzyme
binding
site

CNH2 C
CH3

O-

O-

- CH2 CH2 O P O P OII

II
O
O

H
C
H

C-C-OH
H

H3C

Thiamine Pyrophosphate
(TPP; Vit B1-derived)

AMP

PYRUVATE (C3)
NAD
Pyruvate

Role of TPP in the

Oxidative DecarboTPP
NADH + H+
dehydrogenase
xylation Reactions
CO2
of CAC
ACETYL-CoA
(C2)
Citrate synthase
(C6) OXALOACETATE
NADH + H+

Malate
dehydrogenase
(C4) MALATE
Fumarase

CITRATE (C6)

H2O

Aconitase
H2O

NAD

ISOCITRATE (C6)
NAD

H2O

E
T
C

ATP
Oxid.
Phospho

(C4) FUMARATE

TPP
NADH + H+
CO2

KETOGLUTARATE (C5)

FADH2

Succinate
dehydrogenase

FAD

(C4) SUCCINATE
CoA

Isocitrate
dehydrogenase

NAD

H2O O2 NADH + H+ TPP

CO2

(ATP)
GTP ADP + Pi

- ketoglutarate
dehydrogenase

SUCCINYL CoA (C4)

Mg+

Succinyl CoA thiokinase

COENZYME: FAD
NH2

H3C
H3C

H3C

Ribitol

N
N

NH
N

Isoalloxacin ring

CH2
From ATP
I
H-C-OH
I
H-C-OH
I
N
H-C-OH O
O
I
II
II
CH2-O-P O P
P O-CH2 N
I
O
O
O

NH2
N
N

OH OH

Flavin Adenine Dinucleotide (FAD)

A riboflavin (Vit B3)-derived coenzyme of
several dehydrogenases involved in
oxidation-reduction reactions.

COENZYME: FAD
ROLE OF FAD IN THE CITRIC ACID CYCLE
Succinate
dehydrogenase

COOH
|
CH2
|
CH2
|
FAD
COOH(C4)
Succinate

FADH2

H -COOH
|
HC
||
CH
|
H -COOH

Fumarate (C4)

COENZYME: FAD
NH2
+ I
H2N = C
I
NH
I
CH2
I
CH2
I
CH2
+
I
H3N C H
I
COO-

Arginine

ROLE OF FAD AND FMN IN

NITRIC OXIDE (NO) SYNTHESIS
NADPH2
+ O2

NADP+
+ H2 O

NO +
Nitric oxide synthase
FAD, FMN, Heme
Tetrahydrobiopterin

NH2
I
C=O
I
NH
I
CH2
I
CH2
I
CH2
+
I
H3N C H
I
COO-

Citrulline

COENZYME:PLP
Reactive aldehyde group
involved in the transfer
of amino groups.

O
O C HH
-

OH

O3PO-CH2

+
N

CH3

Pyridoxal
Phosphate
A Vit. B6-derived coenzyme involved in
carbohydrate, amino acid and
neurotransmitter synthesis.

COENZYME: PLP
ROLE OF PLP IN CARBOHYDRATE METABOLISM:
GLYCOGENOLYSIS
Glycogen
chain

HO
OH

O
OH
H

HO
O

OH

OH

O-PO
OH

Glucose 1-P

O
OH

HO
=
3

HO

O
OH

OH

HO
O

OH

H OH

OH

Glygogen
phosphorylase

PLP

OH

OH
H

Pi

HO

O
HO

OH
H

OH

O
OH

OH

HO
O

O
OH
H

Remaining glycogen

H
OH

OH

COENZYME: PLP
ROLE OF PLP AS A COENZYME IN AMINO
ACID METABOLISM: HEME SYNTHESIS
Glycine +Succinyl CoA
-Aminolevulenate
synthase

PLP

-Aminolevulenic acid
(ALA)
several
reactions

Heme
(Fe protoporphyrin IX)

COENZYME: PLP
ROLE OF PLP IN AMINO ACID METABOLISM:
HISTAMINE SYNTHESIS
H
|
CH2 C COO|
NH3

Histidine
PL
P

Histidine
decarboxylase

CO2
CH2 CH2 NH3

HISTAMINE

COENZYME: PLP
ROLE OF PLP IN AMINO ACID METABOLISM:
TRANSAMINATION
COO|
Alanine
CH2
aminotransferase
|
(transaminase)
NH3+
CH2
|
|
O
+
- +
CH3 C C COO
C=O
||
|
|
CH3 C COOPLP
H
COOPyruvate
Alanine
-Ketoglutarate

COO|
CH2
|
CH2
|
H C NH3+
|
COOGlutamate

ENZYME COFACTORS
Cofactor

Enzyme

B. Inorganic (Metal ions

or iron- sulfur clusters)
Zn+2

Carbonic anhydrase, Alcohol dehydrogenase, Carboxypeptidases A & B

Cu+2

Cytochrome oxidase

Mn+2

Arginase, Ribonucleotide reductase

Mg+2

Hexokinase, Pyruvate kinase, Glucose 6phosphatase

Ni+2

Urease

Mo

Nitrate reductase

Se

Glutathione peroxidase

Mn+2

Superoxide dismutase

K+

Propionyl CoA carboxylase

ENZYME COFACTORS: Mg+2

O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 OH
|
H

Glucose

Hexokinase
Glucokinase

Mg+2
ATP

ADP
+ Pi

O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O P
|
H

Glucose 6-PO4

GLYCOLYSIS
ENZYME COFACTORS: K+
O
||
C1 O |
C2 O ~ P
|
H C3
|
H

Pyruvate kinase

Phosphoenol Pyruvate
(PEP)

K+
ADP

ATP

COO|
C2 = O
|
CH3
Pyruvate

G0 = - 6.1 kcal/mole

ENZYME COFACTORS: Zn+

Carbonic anhydrase

CO2 + H2O

H2CO3
Zn+2

METALLOENZYMES
Enzymes that require
a metal in their
composition

SUBSTRATE
The molecule acted upon
by the enzyme
to form a
product

ATP AS A CO-SUBSTRATE
O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 OH
|
H

Glucose

O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|

Hexokinase/
Glucokinase

Mg+2

ATP

ADP

G0 = - 4.0 kcal/mole

H - C6 - O P
|
H

Glucose 6-Phosphate

ACTIVE SITE OF THE

ENZYME

LYSOZOME: ACTIVE SITE

CHYMOTRYPSIN:ACTIVE SITE

His 57

Ser 195

ALLOSTERIC SITE

Substrate

Substrate sites

Enzyme

Allosteric site

REGULATORY ENZYME
The enzyme that catalyzes the
rate-limiting or committed
step of a metabolic
pathway.

REGULATORY ENZYME
Phosphofructokinase I
H
|
H - C1 - OH
|
C2 = O
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O P
|
H

H
|
H - C1 - O - P
|
ATP
ADP + Pi
C2 = O
|
OH - C3 - H
|
H - C4 - OH
Phosphofructokinase I
|
H - C5 - OH
+
|
ATP
H - C6 - O P
AMP
Citrate
|
F 2,6 bisPO4
+
H
H

Fructose 6phosphate

Glycolysis

Fructose 1,6bisphosphate

REGULATORY ENZYME
Acetyl CoA Carboxylase
Citrate
Insulin
High CHO
Low Fat
High Prot.

O
||
CH3 C S CoA

CO2 ACETYL CoA

(HCO3-)

Acetyl CoA carboxylase

Malonyl CoA
Palmitoyl CoA
Epinephrine
Glucagon
High Fat
Fasting

ATP
ADP + Pi
H2O

O
O
\
||
C CH2 C S CoA
//
MALONYL CoA
O

De Novo Synthesis of Fatty Acids

REGULATORY ENZYME
HMG Coa Reductase
Insulin, T3
Glucocorticoids

Cholesterol
Bile acids
Mevalonate

Glucagon
Statins

O
II
+
OC
I
HMG CoA reductase
CH2
I
OH C CH2
I
C S CoA NADPH
NADPH
CoA
II
+ H+
O

HMG CoA

C1OO|
C2H2
|
HO C3 CH3
|
C4H2
|
C5H2OH

Mevalonate

Cholesterol Synthesis

REGULATORY ENZYME
Glucose 6-Phosphate
Dehydrogenase
H
|
C1 = O
|
H C2 OH
|
HO C 3 H
|
H C4 OH
|
H C 5 OH
|
C6H2OPO32-

Glucose 6-phosphate

NADP

NADPH
+ H+

Glucose 6-phosphate
dehydrogenase

O
||
C1
|
H C2 OH
|
HO C3 H
|
H C4 OH
|
H C5
|
C6H2OPO32-

6-phosphogluconolactone

Pentose Phosphate Pathway

INTRACELLULAR LOCATION OF SOME

IMPORTANT BIOCHEMICAL PATHWAYS

ISOENZYME
Different structural forms of an
enzyme which catalyze the same
chemical reactions act on the
same substrate(s) and produce the
same product(s) but exhibit differing
degrees of efficiency.
Different isoenzymes are expressed in
specific tissues of the body.

ISOENZYMES OF LACTATE
DEHYDROGENASE
Lactate dehydrogenase
(LDH) catalyzes the
reversible conversion of
pyruvate to lactate.
Tetramer consisting of 2
subunits: M (found in
skeletal muscles and
liver) & H (heart).
5 distinct isoenzyme forms
(from combination of M &
H isozymes).
An increase of H4 in the blood
indicates tissue damage
as in heart attack.
Enzymes can therefore serve
as markers for disease.

SIX MAJOR CLASSES OF ENZYMES (IUBMB*, 1964)

CLASS

EXAMPLE

Oxidoreductases

Dehydrogenases, Oxidases, Reductases,

Peroxidases, Catalases, Oxygenases,
Hydroxylases

Transferases

Transaldolase and Transketolase; Acyl, methyl

glucosyl, and phosphoryltransferases,
Kinases, Phosphomutases, Transaminases

Hydrolases

Esterases, Glycosidases, Peptidases,

Phosphatases, Thiolases, Phospholipases,
Amidases, Deaminases, Ribonucleases

Lyases

Decarboxylases, Aldolases, Hydratases,

Dehydratases, Synthases, Lyases

Isomerases

Epimerases, Isomerases, Mutases, Racemases

Ligases

Synthetases, Carboxylases

*International Union of Biochemistry and Molecular Biology; classification is

based on the reactions enzymes catalyze; each class is divided into subclasses.

OXIDOREDUCTASES
Transfer of electrons and hydrogen
atoms from donors (or reductants,
hence oxidized to acceptors (or
oxidants, hence reduced).
COO
|
HO C H + NAD+
|
CH3
-

L-Lactate

Lactate
dehydrogenase

COO|
C = O + NADH + H+
|
CH3

Pyruvate

TRANSFERASES
Transfer functional groups (like C-, N-, or
P-) from donors to acceptors; utilize 2
substrates to produce 2 products.
COOAlanine
COOCOOtransaminase
|
|
|
H3N C H + C = O
C = O + H3N C O
|
|
|
|
PLP
CH3
(CH2)2
CH3
(CH2)2
L-Alanine
|
Pyruvate
|
(amino acid)
(keto acid)
COOCOOsubstrate -Ketoglutarate
product
L-Glutamate
(keto acid)
(amino acid)
substrate
product

TRANSFERASES

VI.
Reactions:
GLYCOLYSIS
Kinase
- transfers the
functional group
phosphate from ATP to an acceptor

O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 OH
|
H

Glucose

Hexokinase
Glucokinase

Mg+2
ATP
(donor)

ADP + Pi
(product)

G0 = - 4.0 kcal/mole

O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O P
|
H

Glucose 6phosphate
(product)

TRANSFERASES
Glycosyltransferase if the transferred
group is a carbohydrate residue
OH
I H
CH-C=C-(CH2)12-CH3
H

0
II
R1-C-N-CH
I H
CH2OH

Galactosyl
transferase

Ceramide
UDP-galact- UDP
ose

Fatty
acid

OH
I H
0
CH-C=C-(CH2)12-CH3
H
II
R1-C-N-CH
I H
O
CH2
O
HOCH2
HO H
OH
H
H

Galactose

H
OH

Cerebroside
(Galactocerebroside;
a glycosphingolipid)

HYDROLASES
Catalyze cleavage of chemical bonds by
addition of H2O, producing 2 products
Phosphate bond
O
O
Pyrophosphatase
||
||
O P ~ O P ~ O- + HOH
|
|
O
OPyrophosphate
(PPi)

O
||
2 HO P O|
O
Phosphate
2 (Pi)

HYDROLASES

Catalyze cleavage of chemical bonds,

producing 2 products
O
O
||
||
CH3-(CH2)12-C-CH2-C-S-CoA
-Ketoacyl CoA

Thiolase
Acetyl CoA

CoA
O
||
CH3-C-S-CoA
CH3-(CH2)12-C-S-CoA
Fatty acyl CoA (2 carbons shorter)

LYASES
Cleave C-C, C-O, C-N bonds by means
other than hydrolysis or oxidation
O O\\ /
C
|
C = O H+
|
CH3
Pyruvate

Pyruvate
decarboxylase

H O
\ //
C
|
CH3

+ O=C=O
Carbon
dioxide
(CO2)
Acetaldehyde

LYASES
Cleave C-C, C-O, C-N bonds by means other
than hydrolysis or oxidation
4
HO

CH2 CH COOI
NH3+

OH

3,4-Dihydroxyphenylalanine (DOPA)
PLP

DOPA decarboxylase

CO2

+
CH2 CH2 NH3
HO
OH

Dopamine

LYASES
Cleave C-C, C-O, C-N bonds by means other
than hydrolysis or oxidation
H
|
CH2 C COO|
NH3

Histidine
PLP

Histidine
decarboxylase

CO2

CH2 CH2 NH3

HISTAMINE

GLYCOLYSIS
LYASES
Catalyze C-C bond cleavage in a reversible reaction
P
P
|
|
O O H
OH OH O
|
||
|
|
|
|
H - C1 C2 C3 - C4 - C5 - C6 - H
|
|
|
|
|
H
OH H
H H
Fructose 1,6-Bisphosphate
O
||
H C1 H
|
H C2 OH
|
H C3 O P
|
H
Glyceraldehyde
3 Phosphate (GADP)

G0 = + 5.73 kcal/mole

Aldolase A

O
||
H C4 O P
|
H C5 OH
|
H C6 OH
|
H
Dihydroacetone
Phosphate (DHAP)

LYASES
Synthase catalyzes a physiologically important
reaction that favors the formation of a C-C bond
C1OO|
C2= O
|
C3H2
H2O
|
C4OOOxaloacetate (C4)
S~CoA
|
C1 = O
|
C2H3
Acetyl CoA (C2)

HS-CoA

Citrate synthase

C1OO|
C2H2
|
HO C3 C4OO|
C5H2
|
C6OOCitrate (C6)

LYASES
Synthase catalyzes a physiologically important
reaction that favors the formation of a C-C bond
CH2OH
I
HC-NH3+
I
COOSerine

H2O
PLP

HS-CH2-CH2-CH-COOI
Cystathionine synthase
NH3+

Homocysteine

S
CH2
CH2
I
I
CH2
H -C-NH3+
I
I
CH-NH3+
COOI
COO-

Cystathione

LYASES
Hydratase add H2O to a susbtrate

C1OO|
H C2
||
C3 H
|
C4OO-

Fumarate

H2O

Fumarase
(or fumarate hydratase

C1OO|
HO C2 H
|
H C3 H
|
C4OO-

Malate

ISOMERASES
Transfer of functional groups or double
bonds within the same molecule
C1OO|
H3N C2 H
|
C3H3

L-Alanine

Alanine
racemase

C1OO|
H C2 NH3
|
C3H3

D-Alanine

ISOMERASES
GLYCOLYSIS
Transfer of functional groups or double
bonds within the same molecule
O
||
H C1 H
|
H C2 OH
|
H C3 O P
|
H

Glyceraldehyde
3 Phosphate (GADP)

Triosephosphate
isomerase

O
||
H C1 O P
|
H C2 OH
|
H C3 OH
|
H

Dihydroacetone
Phosphate (DHAP)

ISOMERASES
Transfer of functional groups or double
bonds within the same molecule
O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O P
|
Glucose H
6-Phosphate
(Aldose)

Aldehyde group
Keto group

ATP

H
ADP
|
H C1 - OH
|

C2 = O
|
Phosphohexoisomerase
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O P
|
H
Fructose 6-Phosphate
(Ketose)

ISOMERASES
Transfer of functional groups or double
bonds within the same molecule
O
||
C1 O|
H C2 OH
|
H C3 O P
|
H

3-Phosphoglycerate

Phosphoglycerate mutase
Mg+2
G0 = + 1.06 kcal/mole

O
||
C1 O|
H C2 O - P
|
H C3 OH
|
H

2-Phosphoglycerate

ISOMERASES
Transfer of functional groups or double
bonds within the same molecule
C1H2OH
I
C2=O
I
HOC3H
I
HC4OH
I
H2C5OPO32-

Epimerase

D- Xylulose 5-phosphate

C1H2OH
I
C2=O
I
HC3OH
I
HC4OH
I
H2C5OPO32-

D- Ribulose 5-phosphate

C-3 Epimers

LIGASES
Catalyze the joining of
substrates in the presence of ATP.

CH3
ATPADP + Pi
Pyruvate carboxylase
|
C=O
|
COO- BiotinPyruvate

CO2

ATP

ADP
+ Pi

COO|
CH2
|
C=O
|
COO-

Oxaloacetate

CHARACTERISTICS OF
ENZYMES
They are not changed by the
reaction they catalyze.

CHARACTERISTICS OF
ENZYMES

They do not change or alter

the equilibrium of the
chemical reaction.

CHARACTERISTICS OF
ENZYMES

They increase reaction

rates by decreasing
the activation
energy.

ENZYMES DECREASE THE

ACTIVATION ENERGY
Reaction Coordinate Diagram
+
+
Transition state, S
+
+
G (uncatalyzed)
+
+ G

Free energy, G

(catalyzed)

Substrates
or
+
Reactants
(e.g. CO2 + H2O)

G
for the
reaction

Products
(H2CO3)

Reaction progress

CHARACTERISTICS OF
ENZYMES
They are highly specific for
the reactants or substrates
they act on and catalyze
only one type
of chemical
reaction.

ENZYME SPECIFICITY
Carbonic
anhydrase

CO2 + H2O

H2CO3

ENZYME SPECIFICITY
Catalase

2 H2O2

2 H2O

ENZYME SPECIFICITY
COO|
HO - C H
|
CH3
L-Lactate
COO|
H - C OH
|
CH3
D-Lactate

Lactate
dehydrogenase

NAD+

NADH+ + H+

COOI
C OH
I
CH3
Pyruvate

ENZYME SPECIFICITY
O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 OH
|
H

Glucose

Glucokinase

Mg+2
ATP

ADP + Pi

O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O P
|
GlucoseH6-phosphate

CHARACTERISTICS OF
ENZYMES
PEPTIDE BOND

H
H
I
I
R C CO NH C R
I
I
NH2
COOH

They are mostly proteins in nature.

ENZYME-SUBSTRATE COMPLEX FORMATION

The First Step in Enzymatic Catalysis

Reaction velocity (V)

Maximal velocity

Substrate concentration [S]

ENZYME-SUBSTRATE COMPLEX
CYTOCHROME P450-CAMPHOR

Camphor

Cytochrome P-450

MODELS OF ENZYMESUBSTRATE COMPLEX

Lock and Key Model
(Emil Fischer, 1894)
Induced Fit Model
(Daniel E. Koshland, Jr,
1958)

LOCK AND KEY MODEL

INDUCED FIT MODEL

KINETICS OF ENZYMECATALYZED REACTIONS

E+S

k1
k-1
Substrate
binding

ES

k2
k-2
Catalytic
step

E+P

MICHAELIS-MENTEN EQUATION
Vmax [S]
Vo =
{Km + [S]}
Vo = Velocity at any time (moles/time)
Vmax = Maximal velocity (or reaction rate)
Km = Michaelis constant for the particular
enzyme under investigation
= (K-1 + K2)/K1
[S] = Substrate concentration (molar)

MICHAELIS-MENTEN
SATURATION CURVE

Zero order

Vmax

A
Km

A = [S] < Km
B = [S] = Vmax/2

Reaction velocity (VO)

Vmax

First order

Substrate concentration [S]

C = [S] > Km
= Vo is maximal
(Vmax)

REPRESENTATION OF AN ENZYME IN
THE PRESENCE OF A SUBSTRATE

S=
E=

[S] < Km

[S] = Vmax/2

[S] > Km

SIGNIFICANCE OF KM
1. It is the substrate concentration at
which half of the active sites of the
enzyme are filled up.
2. It is an inverse measure of the affinity
of the substrate for the enzyme:
a. The lower the Km, the higher is
the
affinity.
b. The higher the Km, the lower is

KM AND PHYSIOLOGICAL
VI. Reactions:
GLYCOLYSIS
UTILIZATION OF GLUCOSE

O
||
C1 - H
|
H - C2 - OH
|
OH - C3 - H
|
H - C4 - OH
|
H - C5 - OH
|
H - C6 - O P
|
mM
H
Glucose
6-

Hexokinase
O
(Extrahepatic cells, RBCs)
||
C1 - H
Glucokinase
|
(Liver, Pancreatic -cells
H - C2 - OH
|
OH - C3 - H
|
Mg+2
H - C4 - OH
|
H - C5 - OH
ATP
ADP + Pi
|
H - C6 OH
Km for hexokinase = 0.1
|
phosphate
H Km for glucokinase = 5 mM

Glucose

LINEWEAVER- BURKE DOUBLE

RECIPROCAL PLOT

1
V

Km
Slope =
Vmax

Intercept 1
on X-axis =
-

Km

1
Intercept on Y-axis =

Vmax

1
[S]

LINEWEAVER-BURKE DOUBLE
RECIPROCAL PLOT:SAMPLE PROLEM

Y intercept

1/V (mM sec-1)-1

55
50
45
4
0
35
30
25

- .12

Km
Vmax

Slope =

20

Y intercept =

15

Vmax

10

X intercept =

I
1
Km

.05

.1

.
.
1/[S]1(mM-1) 2
5

.25

.
3

LINEWEAVER-BURKE DOUBLE
RECIPROCAL PLOT: SAMPLE PROBLEM
Y intercept =

X intercept =

Vmax

Km

Vmax =

Y intercept
Vmax =

Km =

1
20

Vmax = 0.05 (mM sec-1)-1

Km =
Km =

1
X intercept

1
.12

8.33 (mM-1)

INHIBITION OF ENZYMATIC
REACTIONS
Reversible
a. Competitive
b. Non-competitive
c. Uncompetitive
Irreversible

REVERSIBLE INHIBITION

COMPETITIVE INHIBITION: LOVASTATIN

REVERSIBLE INHIBITON
Inhibitor Type
Competitive
Inhibitor

Noncompetitive
Inhibitor

Uncompetitive
Inhibitor

Binding Site on Enzyme

Kinetic Effect

Inhibitor binds specifically at active or

catalytic site, where it competes with
substrate for binding; inhibition is reversed
by increasing substrate concentration.

Vmax unchanged;
Km increased to
reach a given
velocity.

Inhibitor binds E or ES other than at the

active or catalytic site; substrate binding
unaltered but ESI complex cannot form due
to structural change in the enzyme
catalytic power no products formed;
inhibition cannot be reversed by increasing
substrate concentration since inhibitor
cannot be driven from the enzyme.

Vmax decreased
proportionately to
inhibitor
concentration; Km
unchanged (since
substrate can still
bind to the
enzyme).

Inhibitor binds only to ES complexes at

locations other than catalytic site; substrate Vmax decreased
binding modifies enzyme structure, making
Km decreased
inhibitor-binding site available; inhibition
cannot be reversed by increasing substrate
concentration; rare in occurrence.

LINEWEAVER-BURKE PLOT FOR

COMPETITIVE INHIBITION
Vmax unchanged by
competitive inhibitors since
increasing substrate
concentration can displace
virtually all competitive
inhibitors bound to the
active site, hence the
identical Y-axis intercepts
of Lineweaver-Burke plots,
with or without inhibitor.
A competitive inhibitor
increases Km for a given
substrate in order to attain
Vmax that were reached in its
absence, hence the differing
X intercepts.

Competitive
inhibitor

1
[V]

Uninhibited
enzyme
1
Vmax

-1
Km

1
[S]

LINEWEAVER-BURKE PLOT FOR

NONCOMPETITIVE INHIBITION
Noncompetitive
inhibitor

Vmax is decreased since a

noncompetitive inhibitor
reduces the concentration of
ES complex that can advance
to reaction products, hence
the differing Y-intercepts.
Km is unchanged since
substrate can still bind to the
enzyme, hence the identical
X intercepts of LineweaverBurke plots, with or without
noncompetitive inhibitor.

1
[V]

Uninhibited
enzyme
1
Vmax

1
Km

1
[S]

LINEWEAVER-BURKE PLOT FOR

UNCOMPETITIVE INHIBITION
Uncompetitive
inhibitor
Uncompetitive inhibitors
decrease both Vmax and
Km, hence the
production of parallel
lines in uninhibited and
inhibited reactions.

1
[V]

Uninhibited
enzyme

1
Vmax

1
Km

1
[S]

LINEWEAVER-BURKE DOUBLE RECIROCAL

PLOT IN THE PRESENCE OF AN INHIBITOR
Type of
Inhibition

Vmax

Same

Noncompetitive
inhibitor
Competitive
inhibitor
Uncompetitiv
inhibitor

Km

Competitive

1
[V]

Same

Noncompetitive

Uninhibited
enzyme

1
Vmax

Uncompetitive
-1
Km

1
[S]

IRREVERSIBLE INHIBITION
Diisopropylphosphofluoridate (DIPF)

A. Normal Reaction of Acetylcholinesterase

O
II
+
H3C-C- O-CH2-CH2-N(CH3)3
Acetylcholine

O
II

+
HO- CH2-CH2-N(CH3)3

OH
I
Enz-Ser

Choline

O
II
O- C CH3
I
Enz-Ser

H2O

+ H3C-C-OAcetate

OH
I
Enz-Ser

A. Reaction with Organophosphorus Inhibitors

OH
I
Enz-Ser +

CH3 O
CH3
I
II
I
H-C-O - P - O-C-OI
I
I
CH3 F
CH3

(Enz acetylcholinesterase)

F- , H +

CH3 O
CH3
I
II
I
H-C-O - P - O-C-O- Inactive
enzyme
I
I
I
CH3 O
CH3
Enz-Ser

IRREVERSIBLE INHIBITION - Penicillin

OH
I
Ser
Penicillin
Glycopeptide
I
-lactam
transpeptidase
C=O
ring
I
H-N
H
I
CH3
S
H-C C
C
CH3
I
C
C N
II
H COO
O

Strained peptide
bond

Bacterial enzyme

I
C=O
I
H-N H
CH3
I
S
H-C C
C
CH3
I
C
O=C N
II H
H COO
O
I
Ser
Glycopeptide
transpeptidase

REPRESENTATIVE DRUGS THAT

INHIBIT SPECIFIC ENZYMES
DRUG

ENZYME TARGET

DISEASE

Amrubicin

Topoisomerase II

CA chemotherapy

Antabuse

Aldehyde
dehydrogenase

Alcoholism

Captopril

Angiotensinconverting enzyme

Hypertension

Celebrex

Cyclooxyenase-2

Arthritis

Digoxin

Na+-K+-ATPase pump

Heart problem

HIV protease

Acquired
Immunodeficiency
Syndrome (AIDS)

Lipitor

HMG CoA reductase

Hypercholesterolemia

Viagra

Phosphodiesterase

Erectile dysfunction

Agenerase

ENZYMES AND PHARMACOTHERAPY: ACE INHIBITORS

Angiotensinogen
(liver, polypeptide,
400 AAs)

Angiotensin I
(decapeptide, 10 AAs)
Renin
(JG cells,
placenta)

Angiotensin
converting
enzyme (ACE)
Angiotensin II
(octapeptide, 8 AAs)

Arteriolar
vasoconstriction

BP
Aldosteronemediated renal
Na+ & H2O
reabsorption

ACE Inhibitor
(Captopril,
Functions/
Amlodipine)
Effects

ENZYMES AND PHARMACOTHERAPY: STATINS

O
OH
O
||
|
||
C CH2 C CH2 C S-CoA
/
|
OCH3

HMG CoA (3-hydroxy-3-methylglutaryl CoA )

2 NADPH + 2H+

HMG CoA reductase

2 NADP+

CoA
O
OH
II
|
O C CH2 C CH2 CH2OH
|
CH3
MEVALONATE (C6)

Statins
Atorvastatin
(Ex. Lipitor)
CHOLESTEROL

ENZYMES AND PHARMACOTHERAPY: VIAGRA

Adenylate cyclase

GTP

cGMP
Pi

Phosphodiesterase

Viagra (Sildenafil
citrate)

GMP

cGMP

Smooth muscle
relaxation
and vasodilation
in penile
blood vessels

SUSTAINED ERECTION

ENZYMES AND PHARMACOTHERAPY:

COX-2 INHIBITORS
PHOSPHOLIPIDS (Cell Membrane)

NSAIDS
(ASA,
Indomethacin)

5-Lipoxygenase
5-HPETE
Peroxidase

Prostaglandin
H2 synthase

Spontaneous

Glu

COX-2

COX-1

Prostaglandin
H2 synthase

Pain and
inflammation

5-HETE

LTA4
Leukotriene
(Leukotriene A4)
synthase
Leukotriene
synthase
Glutathione
(Glu-Gly-Cys)
LTC4
LTD4

Phospholipase A2

Arachidonic acid (C20:4)

(Eicosatetraenoic Acid)

-NSAIDS
-Selective
COX-2
inhibitors
(Ex. Celebrex)

Cyclooxygenase
PGG2
(Prostaglandin G2)
Peroxidase

LTB4

PGI2
Prostacyclin
(Prostacyclin) synthase

LTE4
Gly

PGE2

PGH2
(Prostaglandin H2)
Thromboxane
synthase

Isomerase
Reductase

EICOSANOID SYNTHESIS

PGF2

TXA2
(ThromboxaneA2)

ENZYMES AND PHARMACOTHERAPY:

ANTABUSE
CH2 CH2 OH
Ethanol
NAD+

Alcohol dehydrogenase

NADH + H+
CH3 - OH
Acetaldehyde
Aldehyde dehydrogenase
_
Acetate
Antabuse
Acetyl CoA

KINETICS FOR AN
ALLOSTERIC ENZYME

REGULATION OF ENZYME
ACTIVITY
Feedback Inhibition
Allosteric (Non-covalent)
Modification
Covalent Modification
Zymogen Activation
Induction or Repression
of Enzyme Synthesis

FEEDBACK INHIBITION
Original Precursor(s)
Enzyme 1

1
Enzyme 2

2
Enzyme 3

3
Enzyme 4

4
Enzyme 5

Final Products

FEEDBACK INHIBITION
Carbamoyl PO4 + Aspartate
Aspartate transcarbamoylase
(ATCase)

Carbamoyl aspartate

series of
reactions

Cytidine triphosphate (CTP)

RNA & DNA

synthesis

Acetyl
CoA

Acetoacetyl
CoA

2N
A
+ DP
2H +
+

2N
A
+ DP
2H H
+

FEEDBACK INHIBITION OF HMG CoA

REDUCTASE BY CHOLESTEROL

HMG CoA

Mevalonic acid + CoA

HMG CoA
reductase

Acetyl
CoA
> 25 steps

Feedback
inhibition

Cholesterol

ALLOSTERIC MODIFICATION
Allosteric modulator (activator or inhibitor)
Binds to regulatory or allosteric site
Conformational change in the
regulatory enzyme
Effect is transmitted to the active site
Change in shape of the active site
Altered activity

ALLOSTERIC MODIFICATION:
Phosphofructokinase I

COVALENT MODIFICATION
ATP Protein ADP
kinase

ENZYME-

ENZYME-

Ser -- OH

HPO4=

H2O

Phosphoprotein
phosphatase

Ser O PO32-

COVALENT MODIFICATION
OF THE ENZYME
2 ATP

2 ADP

Phosphorylase
kinase

P
Phosphoprotein
2 Pphosphatase 2 H2O
ATP
and/or
G6P

AMP

Glucose

P
Phosphorylase b
(inactive)

Phosphorylase a
(active)

Glycogen phosphorylase

COVALENT MODIFICATION: PYRUVATE

DEHYDROGENASE
ATP

Pyruvate
dehydrogenase
kinase

Pyruvate
dehydrogenase

ADP

Pyruvate
dehydrogenase

(active)

(inactive)

Pi

Pyruvate
dehydrogenase
phosphatase

H2O

MAMMALIAN ENZYMES WHOSE CATALYTIC ACTIVITY IS ALTERED

BY COVALENT PHOSPHORYLATION-DEPHOSPHORYLATION

ENZYMES

Low activity

High activity

Acetyl CoA Carboxylase

EP

Glycogen synthase

EP

Pyruvate dehydrogenase

EP

HMG CoA reductase

EP

Glycogen phosphorylase

EP

Citrate lyase

EP

Phosphorylase b kinase

EP

HMG CoA reductase kinase

EP

E = Dephosphorylated

EP = Phosphoenzyme

ZYMOGEN ACTIVATION
Chymotrypsinogen

ZYMOGEN ACTIVATION:
BLOOD COAGULATION
Clotting Factors
Ca+2
Prothrombin

Thrombin

Fibrinogen

Fibrin

Some of the processes

involved in blood clotting

INDUCTION OR REPRESSION
OF ENZYME SYNTHESIS
Blood glucose Blood glucose levels
levels
(Starvation)
(Well-fed state)
Insulin

Synthesis of
key enzymes involved
in glucose degradation

Glucagon

Synthesis of
key enzymes involved
in glucose synthesis

FACTORS AFFECTING
ENZYME ACTIVITY
Temperature
pH
Substrate
concentration
Co-factors

Reaction velocity (Vo)

EFFECT OF TEMPERATURE
Optimum T

Increasing
enzyme
activity

Heat
inactivation
of the
enzyme

10

20

30

40

50

60

Temperature (oC)

70

80

EFFECT OF pH
Optimum
pH

OPTIMUM pH OF VARIOUS
ENZYMES

EFFECT OF CO-FACTORS:
Chlorides, Bromides, Iodides
Cofactors increase the rate of
enzyme-catalyzed reactions

EFFECT OF SUBSTRATE
CONCENTRATION

Reaction velocity (V)

Vmax

Substrate concentration [S]

ENZYMES IN CLINICAL DIAGNOSIS

CARDIAC ENZYMES AS MARKERS FOR

MYOCARDIAL INFARCTION (MI)

Aspartate
aminotransferase

QUESTION 1
Which of the following is TRUE when a substrate
concentration equals km in an enzyme-catalyzed
reaction?
A. A few of the enzyme molecules are present
as ES complex.
B. Majority of the enzyme molecules are present
as ES complex.
C. Half of the enzyme molecules are present as
ES complex.
D. All of the enzyme molecules are present as
ES complex.

QUESTION 1
Which of the following is TRUE when a substrate
concentration equals km in an enzyme-catalyzed
reaction?
A. A few of the enzyme molecules are present
as ES complex.
B. Majority of the enzyme molecules are present
as ES complex.
C. Half of the enzyme molecules are present as
ES complex.
D. All of the enzyme molecules are present as
ES complex.

QUESTION 2
Competitive inhibition can be relieved by
increasing which of the following?
A. Enzyme concentration
B. Inhibitor concentration
C. Enzyme-substrate concentration
D. Substrate concentration

QUESTION 2
Competitive inhibition can be relieved by
increasing which of the following?
A. Enzyme concentration
B. Inhibitor concentration
C. Enzyme-substrate concentration
D. Substrate concentration

QUESTION 3
Which of the following enzymes requires
biotin as a coenzyme?
A. PEP carboxykinase
B. Pyruvate carboxylase
C. Phosphofructokinase I
D. Pyruvate dehydrogenase

QUESTION 3
Which of the following enzymes requires
biotin as a coenzyme?
A. PEP carboxykinase
B. Pyruvate carboxylase
C. Phosphofructokinase I
D. Pyruvate dehydrogenase

QUESTION 4
An enzyme with a low Km indicates
which of the following?
A. High affinity for the substrate.
B. Requires increased amount of
substrate to become saturated.
C. Vmax can be reached at high
substrate concentration.
D. Less enzyme-substrate complexes
are formed.

QUESTION 4
An enzyme with a low Km indicates
which of the following?
A. High affinity for the substrate.
B. Requires increased amount of
substrate to become saturated.
C. Vmax can be reached at high
substrate concentration.
D. Less enzyme-substrate complexes
are formed.

QUESTION 5
Enzymes can be regulated when
phosphorylated. This type of regulation
is called:
A. Allosteric control
B. Feed back regulation
C. Covalent modification
D. Enzyme induction

QUESTION 5
Enzymes can be regulated when
phosphorylated. This type of regulation
is called:
A. Allosteric control
B. Feed back regulation
C. Covalent modification
D. Enzyme induction

REFERENCES
Lehningers Principles of Biochemistry, Nelson,
D.L., Cox, M.M., 5th ed., pp. 183-220.
Harpers Illustrated Biochemistry, Murray, R. K.,
et. al., 29th ed., pp. 62-82.
Biochemistry with Clinical Correlations, Devlin, M.
T., 7th ed., pp. 378-421.
Biochemistry, Lippincotts Illustrated Reviews,
Champe, P.C., Harvey, R.A., 4th ed., pp. 53- 67.
Principles of Biochemistry, Horton, H.R., et al.,
4th ed., pp. 129-156.
Marks Basic Medical Biochemistry: A Clinical
Approach, Lieberman, M., Marks, A.D., 3rd ed.,
pp. 116-154.

THANK YOU

THANK
YOU
THANK YOU