Transcript File

FIRST BIOCHEMISTRY EXAM
Tuesday 25/10/2016
10-11
40 MCQs.
Location : 102, 105, 106, 301, 302
The Behavior of Proteins: Enzymes,
Mechanisms, and Control
General theory of enzyme action, by
Leonor Michaelis and Maud Menten in 1913
Leonor Michaelis, German
1875–1949
Maud Menten, Canadian
1879–1960
.
Learning Objectives
1. What Is the Michaelis–Menten Approach to Enzyme
Kinetics?
2. How Do Enzymatic Reactions Respond to Inhibitors?
3. Does the Michaelis–Menten Model Describe the Behavior of
Allosteric Enzymes ?
4. What Are the Models for the Behavior of Allosteric Enzymes?
5. What is Lineweaver –Burk plot ?
5 . How Does Phosphorylation of Specific Residues Regulate
Enzyme Activity?
6. What Are Zymogens, and How Do They Control Enzyme Activity?
7. What Are Coenzymes and Cofactors ?
8. What Are Isoenzymes & what is their clinical application.
They postulated that the enzyme first combines
reversibly with its substrate to form an
enzyme-substrate complex in a relatively fast
reversible step:
The ES complex then breaks down in a slower
second step to yield the free enzyme and the
reaction product P:
Michaelis-Menten Model
2
V
=
V max [S]
KM + [S]
Michaelis-Menten
equation
Michaelis-Menten Model
When [S]= KM, the equation reduces to –
V=
Vmax [S]
KM + [S]
2
=
Vmax [S]
[S] + [S]
=
Vmax
2
Michaelis-Menten Model
– It is difficult to determine Vmax experimentally
– The equation for a hyperbola (E saturation curve)
V=
Vmax [S]
KM + [S]
(an equation for a hyperbola)
– Can be transformed into the equation for a
straight line by taking the reciprocal of each side
1
V
=
1
V
=
KM + [S]
V max [S]
KM
V max [S]
=
+
KM
V max [S]
1
V max
+
[S]
V max [S]
The physiological consequence of KM is illustrated
by the sensitivity of some individuals to ethanol.
Such persons exhibit facial flushing and rapid
heart rate (tachycardia) after ingesting even small
amounts of alcohol.
In the liver, alcohol dehydrogenase converts
ethanol into acetaldehyde
Normally, the acetaldehyde, which is the cause
of the symptoms when present at high
concentrations, is processed to acetate by
acetaldehyde dehydrogenase.
Most people have two forms of the acetaldehyde
dehydrogenase, a low KM mitochondrial form
and a high KM cytosolic form. In susceptible
persons, the mitochondrial enzyme is less active
due to the substitution of a single amino acid,
and acetaldehyde is processed only by the
cytosolic enzyme. Because this enzyme has a high
KM, less acetaldehyde is converted into acetate;
excess acetaldehyde escapes into the blood and
accounts for the physiological effects.
Lineweaver-Burk Plot
– which has the form y = mx + b, and is the formula
for a straight line
(linearization)
1
V
KM
=
Vmax
y
=
m
•
1
[S]
+
•
x
+
1
Vmax
b
– a plot of 1/V versus 1/[S] will give a straight line
with slope of KM/Vmax and y intercept of 1/Vmax
– such a plot is known as a Lineweaver-Burk double
reciprocal plot
Lineweaver-Burk Plot
– KM is the dissociation constant for ES; the greater the
value of KM, the less tightly S is bound (less affinity)
to E
KM
slope =
Vmax
x intercept =
-1
KM
1
V
y intercept =
1
Vmax
1
[S]
The units KM are units of [S] that is M or mM
Turnover number
• Turnover number (kcat): is the moles of S
converted to P per mole of E per second.
–Unit of kcat is mol S*mol E-1*s-1
–The higher Kcat value the higher the
reactivity of the E
Turnover Numbers and KM
Values for some typical enzymes
Turnover numbr
KM
-1 -1
-1
[(mol S)•(mol E) •s ] (mmol•liter )
Enzyme
Function
Catalase
Conversion of
H 2O 2 to H2O + O2
4 x 10
Carbonic
anhydrase
Hydration of CO2
1 x 106
7
25
12
4
-2
Acetylcholin- Regeneration
esterase
of acetylcholine
1.4 x 10
Chymotrypsin Proteolytic enzyme
1.9.x 10 2
6.6 x 10
Lysozyme
0.5
6 x 10
Hydrolysis of
bacterial cell wall
polysaccharides
9.5 x 10
-1
-3
Enzyme Inhibition
• Reversible inhibitor: a substance that binds to an
enzyme to inhibit it, but can be released
– competitive inhibitor: binds to the active (catalytic)
site and blocks access to it by substrate
– noncompetitive inhibitor: binds to a site other than
the active site; inhibits the enzyme by changing its
conformation
• Irreversible inhibitor: a substance that causes
inhibition that cannot be reversed
– usually involves formation or breaking of covalent
bonds to or on the enzyme
– Cyanides, Penicillin, Heavy metals, Nerve gas
competitive inhibitor
A competitive inhibitor diminishes the rate of
catalysis by reducing the proportion of enzyme
molecules bound to a substrate. At any given
inhibitor concentration, competitive inhibition
can be relieved by increasing the substrate
concentration. Under these conditions, the
substrate "outcompetes" the inhibitor for the
active site.
Methotrexate is a structural analog of
tetrahydrofolate, a coenzyme for the enzyme
dihydrofolate reductase, which plays a role in the
biosynthesis of purines and pyrimidines .
It binds to dihydrofolate reductase 1000-fold more
tightly than the natural substrate and inhibits
nucleotide base synthesis. It is used to treat cancer.
The cofactor tetrahydrofolate and its structural analog
methotrexate. Regions with
structural differences are shown in red.
noncompetitive inhibitor
In noncompetitive inhibition, which also is reversible, the
inhibitor and substrate can bind simultaneously to an
enzyme molecule at different binding sites . A
noncompetitive inhibitor acts by decreasing the
turnover number rather than by diminishing the
proportion of enzyme molecules that are bound to
substrate. Noncompetitive inhibition, in
contrast with competitive inhibition, cannot be overcome
by increasing the substrate concentration.
Competitive Vs noncompetitive
Inhibition
Competitive Inhibition Illustrated on a Double-Reciprocal Plot. A
double-reciprocal plot of enzyme kinetics in the presence ( I )
and absence ( I ) of a competitive inhibitor illustrates that the
inhibitor has no effect on V max but increases K M.
Noncompetitive Inhibition
Illustrated on a Double-Reciprocal Plot. A double-reciprocal plo
of enzyme kinetics in the presence ( I ) and absence ( I ) of a
noncompetitive inhibitor shows that K M is unaltered and V
max is decreased.
Irreversible inhibitor
Penicillin acts by covalently modifying the enzyme
transpeptidase, thereby preventing the
synthesis of bacterial cell walls and thus killing
the bacteria .
Aspirin acts by covalently modifying the
enzyme cyclooxygenase, reducing the synthesis
of inflammatory signals.
Structure of Penicillin
Formation of a Penicilloyl-Enzyme Complex.
Chymotrypsin Inhibition
Treatment with organofluorophosphates such as
diisopropylphosphofluoridate (DIPF) was found to
inactivate the enzyme irreversibly .
Despite the fact that the enzyme possesses 28
serine residues, only one, serine 195, was
modified, resulting in a total loss of enzyme
activity. This chemical modification reaction
suggested that this unusually reactive serine
residue plays a central role in the catalytic
mechanism of chymotrypsin.
An Unusually Reactive Serine in Chymotrypsin.
Chymotrypsin is inactivated by treatment with
diisopropylphosphofluoridate (DIPF), which reacts only
with serine 195 among 28 possible serine residues
Enzyme regulation
Enzyme activity can be regulated by several ways:
1. Covalent modification (Phosphorylation)
The side chain -OH groups of Ser, Thr, and Tyr can form
phosphate esters
phosphorylation by ATP can convert an inactive
precursor into an active enzyme
 Protein kinases phosphorylate enzymes
 Protein phosphatases remove phosphate
groups
Enzyme regulation
Covalent modification (Phosphorylation)
2. Noncovalent modification
(Allosteric regulation)
Using allosteric effectors (activator
and allosteric inhibitor )
3. Control amount of enzyme:
enzyme synthesis: gene regulation
enzyme degradation
4. Zymogen : an inactive precursor of an enzyme; cleavage of
one or more covalent bonds transforms it into the active
enzyme
– Involved in blood clotting and digestion
– Example chymotrypsinogen
 Chymotrypsinogen
– synthesized and stored in the pancreas
– a single polypeptide chain of 245 amino acid
residues cross linked by five disulfide (-S-S-) bonds
– enzyme trypsin cleaves it to give chymotrypsin
Secretion of Zymogens by an Acinar Cell of the
Pancreas
Proteolytic Activation of Chymotrypsinogen. The three chains of αchymotrypsin are linked by two interchain disulfide bonds
Zymogen Activation by Proteolytic Cleavage
Active enzymes are shown in yellow
Blood-Clotting Cascade
Many Enzymes Require Cofactors for Activity
COENZYMES……Cofactors
Enzyme cofactors
Metal
Enzyme
Zn2+
Carbonic anhydrase
Zn2+
Carboxypeptidase
Mg2+
Hexokinase
Ni2+
Urease
Se
Glutathione peroxidase
Mn2+
Superoxide dismutase
Cu2
Cytochrome oxidase
ISOENZYMES
Isozymes or isoenzymes, are enzymes that differ
in amino acid sequence yet catalyze the same
reaction. Usually, these enzymes display
different kinetic parameters, such as K M, or
different regulatory properties.
They are encoded by different genetic loci,
which usually arise through gene duplication
and divergence.
ISOENZYMES
The existence of isozymes permits the fine-tuning of
metabolism to meet the particular needs of a
given tissue or developmental stage. Consider the
example of lactate dehydrogenase (LDH), an
enzyme that functions in anaerobic glucose
metabolism and glucose synthesis. Human
beings have two isozymic polypeptide chains for
this enzyme: the H isozyme highly expressed in
heart and the M isozyme found in skeletal
muscle. The amino acid sequences are 75%
identical.
The functional enzyme is tetrameric, and many
different combinations of the two subunits are
possible. The H4 isozyme, found in the heart, has
a higher affinity for substrates than does the M4
isozyme. The two isozymes also differ
in that high levels of pyruvate allosterically inhibit
the H4 but not the M4 isozyme. The other
combinations, such as H3M, have intermediate
properties depending on the ratio of the two
kinds of chains.
ISOENZYMES……… LDH
• END
• Chapter 7