Download Module 13 Enzymes and Vitamins Lecture 34 Enzymes

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Module 13 Enzymes and Vitamins
Lecture 34 Enzymes
13.1 Introduction
Enzymes are proteins that catalyze body’s chemical reactions. They can increase the rate
of reaction as much 106 by reducing the activation energy without affecting the
equilibrium. Since the enzymes are proteins, they are made up of amino acids linked
together by peptide bonds. The reactant of an enzyme-catalyzed reaction is called as
substrate (Figure 1). The specificity of the enzyme for its substrate is known as molecular
recognition.
S
P
active site
S
E
E + S
P
E
E
E
E + P
EP
ES
Figure 1. Enzyme-catalyzed reactions.
13. 2 Active Site
Active site of an enzyme is generally a hollow on the protein surface where the substrate
can bind (Figure 2). The substrate is generally bound to amino acids present in the
binding site by
H-Bond
Active site
HO
Ser
v. d, w
intraction
Substrate
Phe
Ionic
bond
CO2-
Enzyme
Asp
Figure 2
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variety of interactions such as van der Waals interaction, H-bonding and ionic bonding.
These interactions are to strong enough to hold the substrate for the reaction to take place,
but weak enough to allow the product to depart once it is produced.
The amino acids present in the active site also assist in the reaction mechanism. For
example, nucleophilic amino acid such as serine is commonly involved in enzymecatalyzed reaction mechanisms and will form covalent bond with the substrate as part of
the reaction mechanism (Figure 3).
X
HO
Substrate
S
O
OH
Product
OH
H2O
Ser
Ser
Ser
Figure 3
13.3 Acetylcholine Hydrolysis
There are many reasons why enzymes catalyze the reactions. In figure 3 we have seen an
example where amino acid in the active site can assist the enzyme mechanism acting as a
nucleophile. Another reason why enzyme acts as catalyst is the binding process itself.
The active site is not ideal shape for the substrate, but when the binding takes place the
shape changes to accommodate the substrate and maximizes the possible interactions of
the substrate with amino acids of the active site. This is called as an induced fit (Figure
4).
Substrate
Substrate
v. d, w
intraction
H-Bond
S
E
HO
Ser
Phe
Ionic
bond
Induced fit
CO2Asp
H
O
Ser
S
CO
Phe
Asp
Figure 4. Induced fit
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E
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These binding process also force the substrate to adopt a specific conformation which is
ideal for the reaction with nucleophile and catalytic amino acids. Furthermore, the
binding between the substrate and enzyme is that important bonds in the substrate may be
strained and weakened, allowing the reaction takes place more easily. For an example,
neurotransmitter acetylcholine is hydrolyzed by the enzyme acetylcholinesterase (Scheme
1). In this process, acetylcholine is bound in the active site such that it is held in position
to undergo reaction with nucleophilic serine OH group. A histidine residue is also
position such a way to act as an acid/base catalyst. H-Bonding between the ester group of
the substrate with a tyrosine residue in the active site also aids to weaken the ester
linkage, allowing it to be cleaved more easily.
O
NMe3
O
O
Acetylcholineesterase
+
OH
Choline
Acetic aicd
Acetylcholine
NMe3
HO
Mechanism
O
O
CH2CH2NMe3
N
O H
NH
O
R
N
O H
Histidine
Serine
(Nucleophile)
O
O
O
NH
Histidine
(Acid Catalyst)
Serine
O
O
O
H
R
N
NH
H2O
+H2O
N
O
NH
O
OH
H
N
O
OH
H N
NH
OH
OH
O H
N
NH
OH
N
Histidine
(Acid catalyst)
Scheme 1. Acetylcholine hydrolysis
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NH
Histidine
(Base Catalyst)
O
O
O
O
O
-ROH
NH
H N
O
Histidine
(Base Catalyst)
Serine
R
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NH
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13.4 Enzyme Inhibitors
Enzyme inhibitors are drugs which inhibit the catalytic activity of enzyme. They can be
classified as competitive and noncompetitive inhibitors. Competitive inhibitors compete
with the substrate for the active site. The greater the concentration of the inhibitor, there
is a less probability for the substrate to enter the active site (Figure 5). On the other hand,
the greater the concentration of the natural substrate, the less efficient is the inhibition.
Inhibitor
S
E
No Reaction
E
Figure 5. Competitive Inhibition
Noncompetitive inhibitors do not compete with the substrate to enter the active site.
Instead, they bind at different region of the enzyme and produces an induced fit, which
changes the shape of the enzyme such as the active site no longer available to the
substrate (Figure 6). Increasing the concentration of the substrate will have no effect on
the level of inhibition.
Active site
E
Induced fit
Active site
unrecogniazle
E
Allosteric site
Inhibitor
Figure 6. Noncompetitive Inhibition
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The binding site of the noncompetitive inhibitors is known as allosteric site. It is often
present in the enzyme at the start of the biosynthesis. The allosteric site binds the final
product of the biosynthesis and provides a feedback control for the biosynthesis pathway.
When the concentration of the biosynthesis product is high, the allosteric site is occupied,
the activity of the enzyme is off. The final biosynthetic product is thus used as a lead
compound for the design of inhibitors that will bind to the allosteric site (Figure 7).
S
P'
P
P'''
P''
E
Figure 7. Allosteric Inhibition
The inhibitors can be reversible or nonreversible. In case of reversible inhibitor, the
inhibitor binds with the enzyme through intermolecular interactions such as H-bond,
ionic bonding and van der Waals interactions. There will be equilibrium between the
enzyme-inhibitor complex and free enzyme and unbound inhibitor. The position of the
equilibrium will depend on the strength of the intermolecular interactions between the
enzyme and inhibitor. On the other hand, in case of non-reversible inhibitors, the
inhibitor may react with enzyme generating a covalent bond. Such bonds are strong and
the enzyme remains permanently blocked making the inhibition irreversible (Figure 8).
X
Covalent link
X
Nu
E
Nu
E
Nu
E
Figure 8. Irreverisble Inhibition
13.5 Enzyme Selectivity
The inhibitor should be selective for the target enzyme in order to avoid the side effects.
There are instances where a particular enzyme may exist in different forms having
different amino acid composition, but identical catalytic reaction. These enzymes are
called isozymes. If the variation is in the binding site, it is possible to design drugs that
will be the isozyme selective.
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Module 13 Enzymes and Vitamins
Lecture 35 Coenzymes and Vitamins
13.6 Introduction
Coenzymes are organic molecules commonly derived from vitamins. Vitamin is a
substance that body can not normally synthesize, but they are required in small amount
for normal body function. In this lecture we will cover niacin, vitamin B1, vitamin B2,
vitamin B6, vitamin B12, vitamin H and vitamin K. For the details of the vitamin A, D and
E, please see the lecture 28.
13.7 Niacin
Nicotinamide adenine dinucleotide (NAD+) is a coenzyme that is used by enzyme as
oxidizing agent for biological oxidations. Since niacin is the portion of coenzyme NAD+,
it is to be added in the diet so that the body can synthesize NAD+ (Figure 1). Figure 2
illustrates an example for the enzyme catalyzed oxidation/reduction reactions using
NAD+/NADH.
CONH2
CONH2
Nicotinamide
Nicotinamide
N
O O
P
O
N
O O
P
O
O
OH OH
N
O
P O
O
NH2
O
N
-
OH OH
NH2
O
O
N
N
Adenine
N
O
-
N
O
P O
O
N
Adenine
N
O
COOH
OH OH
OH OH
D-Ribose
N
D-Ribose
NAD+
Niacin
NADH
Oxidizing Agent
Reducing Agent
Figure 1
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a basic group of amino acid chain
O H
an acidic group of amino acid chain
O H
O
:B-Enzyme
H-B-Enzyme
R
H
N
R
N
R
NADH
NAD+
R
CONH2
Reduction
CONH2
N
R
H-B-Enzyme
R
H
R
CONH2
Oxidation
O
:B-Enzyme
NAD+
H
H
CONH2
..
N
R
NADH
Figure 2
13.8 Vitamin B2
Riboflavin (flavin plus ribitol) is known as vitamin B2. It is required to synthesize
coenzyme flavin adenine dinucleotide (FAD) which is used as oxidizing agent in
enzyme catalyzed oxidation reactions. For example, FAD is used by succinate
dehydrogenase to oxidize succinate to fumarate (Scheme 1).
-
CO2
O2C
-
succinate
dehydrogenase
+
FAD
-
O2C
CO2-
+ FADH2
NH2
N
H
H
H
H
H
ribotol
Riboflavin
Me
flavin
Me
N
O
O
N
N
P
P O
OO O
O
O
H
OH OH
OH
R
OH
N
Me
O
N
OH
+ Sred
H
NH
N
N
N
O Me
O
NH
FAD
N
O
Me
Me
N
H
H
N
O
FADH2
FAD
Scheme 1
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R
N
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O
NH
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13.9 Vitamin B1
Thiamine is known as vitamin B1. It is required to form the coenzyme thiamine
pyrophosphate (TPP). TPP is required by enzymes that catalyze the transfer of a two
carbon fragment from one species to another. For example, pyruvate decarboxylase
enzyme requires TPP for the decarboxylation of pyruvate and transfer the remaining twocarbon fragment to a proton to afford acetaldehyde (Scheme 2).
NH2
N
N
N
Me
S
O -O O
O
P
P O
O
O
-
Me
Thiamine pyrophosphate (TPP)
O
-
O
O
pyruvate decarboxylate
+
H
H
+
CO2
TPP
O
pyruvate
Scheme 2
13.10 Vitamin H
Biotin is known as vitamin H, which is synthesized by bacteria the live in intestines.
Thus, biotin does not have to be incorporated in diet and the deficiencies are generally
rare. Biotin is the coenzyme required by enzymes that catalyze carboxylation of a carbon
adjacent to a carbonyl group. For example, acetyl-CoA carboxylate converts acetyl-CoA
into malonyl-CoA. HCO3- is used as the source of carboxyl group which is attached to
the substrate (Scheme 3). The reaction is also required ATP and Mg2+.
O
HN
NH
COOH
S
Biotin
O
O
Acetyl-CoA Carboxylate
-
O2C
-
SCoA + HCO3 + ATP
Mg2+
SCoA + ADP +
Biotin
Scheme 3
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PO(OH)3
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13.11 Vitamin B6
The coenzyme pyridoxal phosphate (PLP) is derived vitamin B6. The deficiency of
vitamin B6 causes anemia. Enzymes that catalyze the reactions of amino acids require
PLP. One of the common examples is the transamination
using enzyme
aminotransferases (Scheme 4).
HOH2C
CHO
CH2OH
OH
2-
O3POH2C
N
Me
H
Pyridoxal phosphate
PLP
N
Me
H
Pyridoxine
Vitamin B6
O
R
+
H3N
CO2-
+
O
-
O
OH
O-
O
α-ketoglutarate
O
PLP
O
R
Enzyme
CO2- +
-
O
O-
O
NH3+
Glutamate
Scheme 4
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13.2 Vitamin B12
Enzymes that catalyze certain rearrangement reactions require coenzyme B12 which is
derived from vitamin B12. In vitamin B12, cyano group is coordinated to cobalt. In
coenzyme B12, this group is replaced by a 5’-deoxyadenosyl group.
Coenzyme B12 is used by enzymes which catalyze reactions in that a group (Y) bonded to
one carbon changes to hydrogen bonded an adjacent carbon (Scheme 5). For example,

methylaspartate is converted into glutamate where CO2- has migrated to the adjacent
carbon.
a coenzyme B12-requring enzyme
H
Y
Y
H
eg.
CO2-
glutamate mutase
CO2
-
NH3+
coenzyme B12
β-methylaspartate
-
O2C
CO2NH3+
Glutamate
Scheme 5
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13.13 Folic Acid
Tetrahydrofolate (THF) is the coenzyme required by enzymes which catalyze the transfer
of a group having a single carbon to their substrates. The one carbon may be methyl
(CH3), methylene (CH2) or formyl (CH) group. The coenzyme THF is required for the
synthesis of bases in RNA and DNA and synthesis of some aromatic amino acids. Folic
acid is the vitamin used for the synthesis of the coenzyme THF. For example,
thymidylate synthase is the enzyme that catalyzes the synthesis of T’s from U’s. This
transformation requires N5,N10-methylene-THF as a coenzyme (Scheme 6).
H2N
N
HN
N
H
N
N
H2N
HN
N
HN
O
N
H
O
HN
O
O
CO2-
HN
CO2-
HN
CO2-
CO2-
Folic Acid
Tetrahydrofolate (THF)
glutamate
H2N
N
H
N
O
N
Me
HN
HN
NHR
5
5
N -methyl-THF
O
H2N
HN
+
O
N
R'
N
H2N
HN
H
N
N
O H2C
H2N
H
N
N
O H2C
NR
10
thymidylate
synthase
NR
dUMP
N5, N10-methylene-THF
P=2'-deoxyribose5-phosphate
H
N
O
N
HC
HN
5
N , N -methylene-THF
N
N
NR
10
N , N -methylenylTHF
O
Me
HN
O
H2N
+
N
R'
HN
H
N
N
NHR
O
dTMP
DHF
Scheme 6
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N
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13.14 Vitamin K
•
Vitamin K is synthesized by intestinal bacteria.
•
Vitamin K is required for proper clotting of blood. In order for blood to clot,
blood-clotting proteins must bind to Ca2+.
•
Vitamin K is synthesized by intestinal bacteria. Thus, deficiency of vitamin K is
rare.
•
Vitamin KH2 is the coenzyme that is derived from vitamin K.
•
The coenzyme vitamin KH2 is required by enzyme that catalyzes the
carboxylation of the γ-carbon of glutamate side chains in proteins, forming γcarboxyglutamates (Scheme 7).
•
The process uses CO2 for the carboxyl group that is introduced in to the glutamate
chains.
•
The protein involved in the blood clotting contains several glutamates near the Nterminal ends.
γ-Carboxyglutamate makes stronger complex with Ca2+ compared to that of
•
glutamate.
O
OH
O
Vitamin K
OH
Vitamin KH2
H
N
H
N
CO2CO2-
Glutamate Side Chain
enzyme
vitamin KH2
CO2
H
N
CO2-
CO2- CO2γ-Carboxyglutamate side chain
CO2-
Ca2+
O
O- -O
Ca2+
O
Calcium Complex
Scheme 7
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