Transcript Document

Enzymes II
Coenzymes, Regulation, Catalytic
Antibodies and Ribozymes
Enzyme:Coenzyme Partners
Allosteric Enzymes
Cellular Regulation of Enzymes
Site-Directed Mutagenesis and
Catalytic Antibodies
Catalytic RNA
1
Enzyme:Coenzyme Partners
Coenzyme
Organic or organometallic molecule that assists an
enzyme.
Prosthetic group
Coenzymes that are covalently linked or
noncovalently bound very tightly to an enzyme
partner.
Vitamins
A group of relatively small, organic molecules
essential for proper growth and development.
2
Cofactors and coenzymes
Some enzymes require a second species to be
present in order to do their job.
For Cofactor type enzymes:
Apoenzyme - protein portion of enzyme
- almost ready to work.
Cofactor
- prosthetic group needed to
‘activate’ the apoenzyme.
- usually a metal ion that holds
protein in the proper shape.
3
Cofactor example
Co2+
non reacting
apoenzyme
Co2+
4
Coenzymes
A second species that temporarily binds to the
apoenzyme in order for it to work.
+
apoenzyme
coenzyme
holoenzyme
5
Vitamins
Thirteen well-identified vitamins that are
classified by their water solubility.
Part of each coenzyme structure is made
from a vitamin.
Coenzyme
NAD+
FAD
Coenzyme A
Vitamin required
niacin
riboflavin
pantothenic acid
6
Water-soluble vitamins
Pantothenic acid
One of the B vitamins. Abundant in many foods. It is
also manufactured by intestinal bacteria.
HO
O OH CH3
|| |
C - CH2 - CH2 - N - C - C - C - CH2OH
|
| |
H
H CH3
|
O
Uses
Undefined role in metabolism of proteins fats and
carbohydrates. Believed to be converted to
coenzyme A.
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Water-soluble vitamins
Niacin - Vitamin B3
Refers to both nicotinic acid and nicotinamide. Found
in fish, meat, milk, cereals.
O
O
||
||
-C-OH
-C-NH2
Uses
N
nicotinic acid
N
nicotinamide
Works as a coenzyme in the release of energy from
nutrients. Precursor to NAD+ and NADP+.
8
Water-soluble vitamins
Riboflavin - Vitamin B2
Found in milk, eggs, leafy green vegetables.
N
N-H
N
N
|
CH2-CH-CH-CH-CH2
|
|
|
Component of H3Cthe coenzymeH C3
FAD. Plays a
role in the
metabolism of
carbohydrates, fats
and respiratory proteins.
O
||
|
|
Uses
H
OH OH OH OH
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Water-soluble vitamins
Thiamine - Vitamin B1
Found in meat, cereals, leafy green veggies.
CH3
|
NH2 H
C=C-CH2CH2OH
|
| +
N
- C-N
|
C-S
H
|
H3CH
N
Uses
Required for many decarboxylation reactions.
Catalyst in carbohydrate metabolism. Enables
pyruvic acid to be absorbed and carbohydrates
to be released. Also plays a role in the synthesis
of nerve-regulating substances.
10
Water-soluble vitamins
Pyridoxine - Vitamin B6
Found in fish, meat, poultry, green leafy vegetables.
R
|
-CH2-OH
HOH3C-
If R = -CH2OH,
If R = -CHO,
If R = -CH2NH2,
Pyridoxine
Pyridoxal
Pyridoxamine
N
Uses.
Plays role in the synthesis of red blood cells and the
use of fats. Required in synthesis and breakdown of
amino acids.
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Water-soluble vitamins
Folic acid - A B vitamin.
Found in meat, cereals, leafy green vegetables,
intestinal bacteria. Stored in liver.
OH
N
H2
C
N
O
COOH
C
NH
N
H
H2 N
Uses
N
N
CH2
CH2
COOH
Synthesis of purines and pyrimidines.
Coenzyme needed for forming body protein and
hemoglobin.
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Water-soluble vitamins
Biotin - A B vitamin.
Found in liver, egg yolks, cheese, peanuts. Synthesized
by intestinal bacteria.
H2
C
S
H2
C
C
H2
COOH
C
H2
Uses
N
N
H
H
O
Involved in carboxylation
and decarboxylation in
metabolism of fats,
carbohydrates, and
proteins.
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Water-soluble vitamins
H2NC=O
Uses
Production of red & white
blood cells. Growth &
maintenance of nerve
tissue.
H2NCH2C
H3C
H3C
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H2NCCH2
O=C-CH2CH2
NH
HC
HO-CH2
CH3
CH3
N
N
CH2
C O
Co+
O
CH2
CH-CH3
O OP
O O
CH2
CN N
N
N
Found in meat, eggs,
dairy products. Most
recently discovered
vitamin.
||
CH2CNH2
||
N
Vitamin B12 - Cobalamin
O
CH2 CH3 CH3
O
NH2
CH3
CH3
CH2 CH2
C O
CH3
CH3
NH2
OH
C C
H H
O
C
H
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Water-soluble vitamins
Vitamin C - ascorbic acid
Found in fresh fruit and vegetables.
OH
O
Uses
HO
O
CH-CH2OH
OH
Formation and maintenance of collagen. Enhances
absorption of iron from foods. Serves as an
antioxidant.
15
Lipid-soluble vitamins
Vitamin A - trans-retinol
Found in liver, egg yolks, green and yellow leafy
vegetables, fruit.
H3C
Uses
CH3
|
-(CH=CH-CH=CH)2-CH2-OH
CH3
CH3
Maintains skin and mucous membranes of oral cavity,
and digestive, respiratory, reproductive, and urinary
tract. Critical for vision.
16
Lipid-soluble vitamins
Vitamin K1 - phylloquinone
Found in leafy vegetables and intestinal bacteria.
O
CH3
H
C C
H2
CH3
C
CH 3
(CH2CH2 C CH2)2 CH 2
H
CH 3
CH2
C
H
CH 3
O
Uses
Essential in blood clotting. Aids in the formation of
prothrombin - the enzyme need to produce fibrin.
17
Lipid-soluble vitamins
Vitamin D - cholecalciferol
Found in liver and fish oils. Produced in body
when exposed to light.
H3C
H3C
Uses
CH3
CH3
CH2
Necessary
for normal
HO
bone
formation
and for retention of calcium and phosphorus.
18
Lipid-soluble vitamins
Vitamin E - tocopherol
Found in vegetable oils, wheat germ, liver and green
leafy vegetables.
H3C
Uses
O
H3C
OH
|
CH3
H3C
H H3C
H
CH3
H3C
Role of this vitamin is not clearly established.
CH3
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Coenzymes
Let’s look at a few coenzymes that are
produced from enzymes.
NAD+
nicotinamide adenine dinucleotide.
FAD
flavin adenine dinucleotide.
Coenzyme A
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NAD+
reactive
site
O
O
O
C
-
P
O
CH2
O
O
O
O
CH2
-
ribose
nicotinamide
N+
O
OH
P
NH2
OH
N
N
O
OH
OH
NH2
N
adenine
N
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FAD
O
H3C
N
H3C
active site
is highlighted
NH
N
N
H
C
H
H
C
OH
H
C
OH
H
C
OH
H
C
H
O
riboflavin
NH2
N
O
O
P
O
O
-
ribose
CH2
N
O
OH
N
adenine
N
OH
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Coenzyme A
phosphorylated
ADP
pantothenate
unit
NH2
O
O H CH3
C-CH2-CH2-N-C-C-C-CH2
H HO CH3
H-N
O
O
N
N
P O P O
O-
O- CH 2
O
N
N
CH2-CH2
S
H
Sulfhydryl
group
O
OH
O P OO23
Allosteric enzymes
The thousands of reactions involved in
metabolism are grouped in sequences.
A
E1
B
E2
C
E3
D
E4
F
E5
P
While the behavior of most of the enzymes
can be explained by Michaelis-Menten
kinetics, there is typically a step that does
not.
This step involves a regulatory enzyme that
controls the rate for the entire sequence.
24
Allosteric enzymes
A number of factors can influence a
regulatory enzyme.
• concentration of the final product(s)
• beginning substrate sequence
• intermediates formed in the pathway
• an external factor like a hormone
• a combination of the above
25
Allosteric enzymes
In many pathways, the first enzyme is often
the major control enzyme.
A
E1
B
E2
C
E3
D
E4
F
E5
P
• It is influenced by the concentration of
the starting material
• It may also be affected by the amount of
final product.
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Methods of enzyme regulation
End product inhibition
Enzyme - substrate reaction is an equilibrium
If product builds up, the reaction slows.
E+S
ES
ES*
EP
E+P
Equilibrium shifts to left
if product starts to build up
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Positive and Negative Effectors
Effectors or modulators
These are molecules that serve to alter
how an enzyme performs.
Positive effectors stimulate an enzyme
Negative effectors inhibit an enzyme
They act by reversible, noncovalent binding
to a site on the enzyme. This alters the
conformation of the active site.
28
Methods of enzyme regulation
Use of allosteric enzyme
Similar to coenzymes.
Example of positive allosterism.
29
Cellular regulation of enzymes
Covalent modification
Reversible, covalent changes to specific
amino acid side chains.
Common alterations
• Phosphorylation of hydroxyl groups in
serine, threonine or tyrosine.
• Attachment of an adenosyl monophosphate
(AMP) to a similar hydroxyl group.
• Reduction of cysteine disulfide bonds.
30
Covalent modification
Enzymes are used to convert the regulatory
enzyme to either an active or inactive form.
Example - control of glycogen phosphorylase
phosphorylase + 2 ATP
phosphorylase + 2 ADP
OH
OP
OH
OP
Specific serine residues in each of two identical dimers of the enzyme
are phosphorylated. The reaction is catalyzed by phosphorylase
kinase. The process can be reversed using a second enzyme,
phosphorylase phosphatase which effects the removal of phosphate.
phosphorylase + 2 H2O
phosphorylase + 2 Pi
OP
OH
OP
OH
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Covalent modification
Other examples
Attachment of AMP to glutamine synthetase
enzyme + ATP
OH
active form
enzyme + PPi
O-AMP
inactive form
Reduction of cysteine disulfide bonds by AH2
enzyme + AH2
enzyme + A
S
S
SH SH
active form
inactive form
32
Cellular regulation of enzymes
Activation of proteolytic cleavage
Some enzymes are initially produced in an
inactive form - zymogen.
A portion of the protein chain must be
removed to make it active - proteolytic
cleavage. This is irreversible.
inactive form
zymogen
active form
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Chymotrypsin
This enzyme is a proteolytic
enzyme.
produced from
chymotrypsinogen.
Initially, -chymotrypsin
is formed is and
subsequently converted to
-chymotrypsin.
In its final form, it consists
of three polypeptides held
together by two interchain
disulfide bonds.
This enzyme cleaves
peptide bonds. It only
works on amino acids
containing an aromatic
ring phenylalanine,
tyrosine & tryptophan.
34
Chymotrypsin
1
245
chymotrypsinogen
(inactive)
trypsin
1
15 16
245
Arg Ile
chymotrypsin
1
13
16
Leu Ile
-chymotrypsin
(active)
Ser - Arg and Thr - Asn
14 15
147 148
146
Tyr
149
Ala
245
-chymotrypsin
(active)
35
Another example
Blood Clotting - formation of fibrin.
Process requires a series of enzymatic steps.
Many of the enzymes are made as inactive
forms to reduce clotting on its own.
Two pathways can be used to start the process.
Extrinsic
- Activated by tissue damage,
outside the blood vessel.
Intrinsic
- Activated by damage within a
blood vessel.
36
Summary of pathways
Extrinsic pathway
Activation
Intrinsic pathway
Activation
XII
VII
XII*
XI
VII complex*
XI*
IX
VII*
IX*
VII complex*
Common pathway
X
X*
prothrombin
fibrin
thrombin
fibrogen
fibrin polmer
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Fibrin
Ribbon model
of fibrin.
38
Drug interactions
Drugs can be administered to alter the
clotting mechanism.
Example: Heparin - an anticoagulant.
Acts by accelerating the action of the existing
inhibitor of thrombin - antithrombin III.
Antithrombin III inhibits activating the
clotting factors that have a reactive serine
residue at their enzymatically active
centers.
39
Heparin interaction
thrombin
antithrombin
40
Heparin interaction
thrombin
serine
antithrombin
inhibited thrombin
lysine sites
heparin
Addition of heparin makes it easier for thrombin to interact
with antithrombin - positive allosteric effect.
41
Cellular regulation of enzymes
Isoenzymes or isozymes
• Enzymes that have similar but not identical
amino acid sequences.
• Each will catalyze the same biochemical
reaction.
• They differ in kinetics - different KM and Vmax
values.
• Use different effectors and forms of
coenzymes.
• Cellular distribution of each form will vary.
42
Regulation by isoenzymes
The best know example is lactate
dehydrogenase (LDH) which catalyzes the
conversion of pyruvate to lactate in muscle
tissue.
It is a tetramer composed of two possible
types of subunits - M and H
These units are made by separate genes and
have similar but different amino acid
sequences.
43
LDH isoenzyme
In skeletal muscle, the predominate form is
M4. This varies for other tissues.
In the heart, the form is H4.
In the liver, every possible combination is
observed: M4, M3H, M2H2, MH3 and H4.
The reasons for isoenzymes are not well
understood but may allow regulation
based on different metabolic patterns.
44
LDH isoenzyme - M4
45
Site-directed mutagenesis
and catalytic antibodies
Studies have shown that catalysts for
biochemical reactions are not limited to
naturally occurring proteins.
Site-directed mutagenesis
Modification of amino acid sequences of
known enzymes and other proteins.
Catalytic antibodies
Production of protein antibodies using
transition-state analogs
46
Site-directed mutagenesis
Using recombinant DNA procedures, it is
possible to modify a gene to use a different
amino acid in a protein sequence.
• Assists in the study of enzyme structure
and activity.
• Allow for the design of new enzymes and
other proteins with desired properties.
• The approach can be used for the design
of new drug therapies.
47
Catalytic antibodies
Protein antibodies function by tightly
binding and neutralizing foreign
substances (antigens).
Pauling proposed that antibodies were
similar to enzymes but bound substrate
molecules in the transition state.
Studies have been conducted to see if an
antibody with enzyme-like activity could
be produced.
48
Catalytic antibodies
An example.
Hydrolysis of the methyl ester of p-nitrobenzoate.
O
O2N
C
O-
H2O
OCH3
O2N
C OCH3
H
O+
H
transition state
The goal was to produce an antibody that bound
the proposed tetrahedral transition state which is
very unstable.
49
Catalytic antibodies
An analog that was easily prepared was
studied.
O
O2N
P
CH 2CH 2CH2CH 2COO -
O-
It was directly injected into animals and
found to result in antibody production.
This antibody catalyzed reaction was found
to obey Michaelis-Menten kinetics and
accelerate rate on the order of 103-106 fold.
50
Catalytic RNA
Since their discovery, it was believed that all
enzymes were proteins.
In 1981 - 1982, two research group reported
results on catalytic RNA.
In 1989, the Nobel Prize in Chemistry was
awarded to Sidney Altman (Yale) and
Thomas Cech (University of Colorado Boulder) for their discovery.
The term ribozyme is now used for RNA
enzymes.
51
Ribonuclease P
This was the first type of catalytic RNA
discovered and is present in all organisms
• Substrates are at least 60 inactive,
precursor forms of tRNA.
• Ribonuclease P acts to remove a segment of
the ribonucleotide, producing mature tRNA.
• The enzyme consists of a small protein
subunit with a molecular weight of 14,000
and an RNA component of 377 nucleotides.
52
A
A
G
A
C
ppp G
Ribonuclease P
U
C
G
C
G
U
G
U
A
C
G
H O-
C
A
C
A
G
G
C
C
A
G
C
A
U
U
A
C
C
C
C
Portion removed
by RNase P
RNase P functions by
hydrolyitc cleavage of
the phosphodiester bond.
G
A
U
G
C
U
C
G
G
G
U
G
C
C
G
A
The enzyme obeys Michaelis-Menten
kinetics, is only needed in small
amounts and must remain in its native
tertiary structure for activity.
G
G
U
G
C
U
A
G
C
G
C
G
U
C
G
G
C
U
U
G
C
A
G
G
C
C
U
C
C
G
G
U
U
A
G
C
C
C
G
C
U
G
C
G
U
A
C
G
C
G
C
G
A
U
G
U
G
C
G
G
C
53
Self-splicing RNA introns
The second example of catalytic RNA
reported involved studies of intron splicing
in Tetrahymena thermophila.
It was observed that for this protozoan,
splicing of an intron in rRNA was
autocatalytic - it cleaved itself.
This splicing cut out an intron sequence of
414 nucleotides which was later processed
to L-19 IVS RNA, lacking 19 nucleotides.
54
Self-splicing RNA introns
5’
upstream
exon
5’
G
OH
..
G
OH
downstream
exon
3’
3’
55
Self-splicing RNA introns
5’
5’
- 19
nucleotides
Spliced
exons
L-19
IVS
RNA
+
414
nucleotides
3’
3’
56
Self-splicing RNA introns
List of activities for catalytic RNA
• Cleavage and rejoining of oligonucleotide
substrates.
• Cleavage of DNA phosphodiester bonds.
• Cleavage of RNA at sequence-specific
sites.
• Hydrolysis of esters.
• Formation of peptide bonds between
amino acids.
57
Significance of ribozymes
Other forms of catalytic RNA continue to be
discovered.
• Small ribozymes have been found as
components of plant RNA viruses.
• The active region of this RNA consists of
only 19 - 30 nucleotides.
• Because of their characteristic shape and
action, they are called “hammerhead”
ribozymes.
58
Hammerhead ribozyme
59