Transcript Zhan-3-Enzyme

Enzymes: Basic Concepts and
Kinetics
Protein catalysts
I. Nomenclature
Each enzyme is assigned two names:
1. Recommended name: short, convenient; common suffix
“ase” attached to the substrate (reactants) or to
the action performed (e.g. proteinase, DNase; lactate
dehydrogenase, adenylyl cyclase); some trivial names, e.g.
pepsin
2. Systematic name: developed by IUBMB ,
6 major classes, each with numerous
subgroups, unambiguous and informative
e.g. D-glyceraldehyde 3-phosphate: NAD
oxidoreductase
II. Properties of enzymes
Protein catalysts that increase the velocity of a
chemical reaction.
1. Active sites:
special pocket or cleft containing aa side
chains that form a 3-D surface
complementary to the substrate.
Common features of active sites
• A 3-D cleft or crevices formed by groups from
different parts of amino acid sequence (Fig 3.1).
• Small part of the total volume of an enzyme.
more amino acids serve as a scaffold, regulatory
sites, sites for interaction with other proteins, or the
channels to bring the substrate to active sites.
• Substrates are bound to enzymes by multiple weak
attractions. Noncovalent interations: electrostatic
interactions, hydrogen bonds, van der Waals forces,
hydrophobic interactions.
Asp101
Asp101
Trp108
Asp52 Glu35
Active site
Catalytic groups
Figure 3.1 Active sites may include distant residues. (A) Ribbon diagram of the
enzyme lysozyme with several components of the active site shown in color. (B) A
schematic representation of the primary structure of lysozyme shows that the active
site is composed of residues that come from different parts of the polypeptide chain.
Figure 3.2 Substrates are bound to enzymes by multiple
weak attractions. (left) The enzyme cytochrome p-450 is
illustrated bound to its substrate camphor. (right) In the active site,
the substrate is surrounded by residues from the enzyme. Note also
the presence of a heme cofactor.
2. Catalytic efficiency
• High efficient: 103 – 108 times faster, each
enzyme molecule can transform 100-1000
substrate molecules to product per second.
• Turnover number: the number of substrate
molecules converted into product by an
enzyme molecule in a unit time when the
enzyme is fully saturated with substrate.
3. Specificity
• High specificity: no side reactions
absolute specificity (only one substrate),
relative specificity (one type of chemical
bond), steric specificity (only one optical
isomer)
Proteolytic enzymes (e.g. proteinases)
Figure 3.3 Enzyme specificity. (A) Trypsin cleaves on the
carboxyl side of arginine and lysine residues, whereas (B) thrombin
cleaves Arg-Gly bonds in particular sequences specifically.
4. Cofactors
•
•
•
•
•
Apoenzyme + cofactor = holoenzyme
No protein cofactors:
Metal ions: Zn2+, or Fe 2+
Organic molecules: coenzymes, derivatives
of vitamins
Holoenzyme: enzyme with its cofactor
Apoenzyme: the protein portion of the
holoenzyme
Prosthetic group: a tightly bound coenzyme
5. Regulation
Enzyme activity can be regulated in the cell:
activated or inhibited
6. Location within the cell
• In specific organelles of the cell
(compartmentalization)
• Isolating the reaction substrate or product
from other competing reactions
• Providing a favorable environment for the
reaction
Fig 3.4 Intracellular
location of some
important biochemical
pathways involved
enzyme-catalyzed
reactions.
III. The mechanism of enzymes action
1. Energy changes occurring during the
reaction
energy barrier (free energy of activation)
chemical reaction:
A ⇌ S‡ ⇌ B
Reactant A to product B through the transition
state S‡ (high energy intermediate)
• Free energy of activation:
The peak of energy in Figure 3.5 is the
difference in free energy (∆G‡) between the
reactant and S‡ ;
Because of the high free energy of
activation, the rates of uncatalyzed chemical
reactions are often slow.
Figure 3.5 Enzymes decrease the activation energy.
• Rate of reaction:
Molecules must overcome the energy barrier
of the transition state.
The rate is determined by the energized
molecules. The lower the ∆G‡ , the faster
the rate.
• Alternate reaction pathway
an enzyme provides alternate reaction
pathway with a lower ∆G‡ , does not change
the ∆G, and equilibrium of the reaction.
Figure 3.6 Enzymes decrease the activation energy.
2. Chemistry of the active site
• Active site: binding substrates and form a
complex molecular machine employing a
diversity of chemical mechanisms to
facilitate the conversion of substrate to
product.
Many factors related to catalytic efficiency
of enzymes.
• Transition-state stabilization:
active site as a flexible molecular template
for the activated transition state
Enzymes stabilize the substrate in its
transition state, and increase the
concentration of the reactive intermediate,
thus accelerates the reaction.
• Other mechanisms: catalytic groups in
active site enhance the formation of the
transition state.
general acid-base catalysis
covalent enzyme-substrate complex
(E + S ⇌ ES → E + P)
Figure 3.7 Lock-and-key model of enzyme-substrate
binding. In this model, the active site of the unbound enzyme is
complementary in shape to the substrate.
Figure 3.8 Induced-fit model of enzyme-substrate
binding. In this model, the enzyme changes shape on substrate
binding. The active site forms a shape complementary to the substrate
only after the substrate has been bound.
Ⅳ. Factors affecting reaction velocity
1. Substrate concentration
•
The velocity or rate : the number of substrate molecules
converted to product per unit time.
The velocity will increase with substrate concentration
until a maximal velocity (Vm) is reached (reflecting the
saturation of binding sites ).
Michaelis-Menten kinetics
E + S ⇌ ES → E + P
V0= Vmax[S]/(Km+[S])
The plot of initial reaction velocity ( V0 ) against
substrate concentration ([S]) is hyperbolic. Some
allosteric enzymes show a sigmoidal curve.
Figure 3.9 Reaction velocity versus substrate concentration
in an enzyme-catalyzed reaction. An enzyme-catalyzed
reaction reaches a maximal velocity.
Figure 3.10 Kinetics for an allosteric enzyme. Allosteric
enzymes display a sigmoidal dependence of reaction velocity on
substrate concentration.
2. Temperature
• Increase of velocity with temperature:
increased number of activated molecules
• Decrease of velocity with higher temperature:
denaturation of protein enzymes
• Optimal temperature
most proteins in human 37ºC
Heat inactivation
of enzyme
V
Temperature, ºC
Figure 3.10 Effect of temperature on
an enzyme-catalyzed reaction
3. pH
• Effect of the pH on the ionization of the active
site: the pH affects reactive groups of both enzyme
and substrate in ionized or unionized state.
• Effect of pH on enzyme denaturation
extremes of pH cause protein denaturation
• The pH optimum varies for different enzymes: the
pH at which maximal enzyme activity is achieved.
pepsin – pH 2, many other enzymes- pH 7.4
Figure 3.11 Effect of pH on enzyme-catalyzed
reactions
V. Michaelis-Menten equation
1. Reaction model: for one substrate molecule
k1
E + S
⇌
k2
ES → E + P
k-1
S – the substrate, E – the enzyme
ES – the enzyme-substrate complex
P – the product
k1, k-1and k2 – the rate constants
2. Michaelis-Menten equation:
Reaction velocity varies with substrate
concentration:
V0= Vmax[S] / (Km+[S])
V0 = initial reaction velocity
Vmax= maximal velocity
[S] = substrate concentration
Km = Michaelis constant = (k-1+ k2)/k1
The assumptions made in deriving the equation
• Relative concentrations of E and S: [S] is much
greater than [E]. The bound substrate is a very
small part of total substrate at any one time.
• Steady-state assumption: [ES] does not change
with time, the rate of formation of ES is equal to
that of the breakdown of ES.
• Initial velocity (V0): the rate measured as soon as
enzyme and substrate are mixed. At that time, [P]
is very small, the rate of back reaction from P to S
can be ignored.
3. Important conclusions about
Michaelis-Menten kinetics
• Characteristics of Km:
a characteristic constant of an enzyme
affinity of the enzyme for that substrate
When Vo=1/2 Vmax, Km=[S]
( small Km: high affinity of the enzyme for
substrate; large Km: low affinity)
• Relationship of velocity to enzyme concentration:
The rate of the reaction is directly proportional to the
enzyme concentrations.
• Order of the reaction:
When [S] << Km, the velocity is proportional to the
[S]---first order
When [S] >>Km, the velocity is constant and equal to
Vm---zero order
V0= Vmax[S] / (Km+[S])
Figure 3.12 Michaelis-Menten kinetics. A plot of the reaction
velocity as a function of the substrate concentration for an enzyme that
obeys Michaelis-Menten kinetics.
• Lineweaver-Burke plot:
If 1/Vo is plotted versus 1/[S], a straight line is
obtained (double-reciprocal plot).
The plot is used to calculate Km and Vmax.
The equation:
1/Vo = Km/Vmax [S] + 1/Vmax
The intercept on the x axis is equal to -1/Km,
The intercept on y axis is equal to 1/Vmax
(Michaelis-Menten equation:
V0= Vmax[S] / (Km+[S]) )
Figure 3.13 A double-reciprocal or Lineweaver-Burk plot.
Ⅵ. Inhibition of enzyme activity
Inhibitors: substances that diminish the
velocity
Reversible and irreversible inhibition
The two most commonly encountered
types of reversible inhibition:
competitive and non-competitive
inhibitions
Figure 3.14 Distinction between a competitive and a
noncompetitive inhibitor.
1. Competitive inhibition
Inhibitors binds to the same site that the
substrate would occupy.
• Effect on Vmax :
The inhibitor effect is reversed by
increasing [S]. The velocity reaches Vmax at
a sufficiently high substrate concentration.
• Effect on Km : apparent Km increased,
More substrate is needed to achieve ½ Vmax.
• Effect on Lineweaver-Burke plot:
The plots of the inhibited and uninhibited
reactions intersect on the y axis at 1/Vmax
(Vmax is unchanged).
Figure 3.15 Reaction of a competitive inhibitor.
Figure 3.16 Kinetics of a competitive inhibitor. As the
concentration of a competitive inhibitor increases, higher concentrations
of substrate are required to attain a particular reaction velocity. The
reaction pathway suggests how sufficiently high concentrations of
substrate can completely relieve competitive inhibition.
Figure 3.17 Competitive inhibition illustrated on a
double-reciprocal plot.
Figure 8.18 Enzyme inhibitors. The cofactor tetrahydrofolate
and its structural analog methotrexate (MTX). Regions with
structural differences are shown in red. MTX inhibits the
dihydrofolate reductase, which plays a role in biosynthesis of
purines and pyrimidines.
2. Non-competitive inhibition
• Inhibitor and substrate bind at different sites on
the enzyme.
• Effect on Vmax :
The inhibitor effect cannot be overcome by
increasing [S]. Thus, it will decrease Vmax of the
reaction.
• Effect on Km : Km remains same
• Effect on Lineweaver-Burke plot:
Vmax decreases, Km is unchanged
Figure 3.19 Reaction of a non-competitive inhibitor.
Vmax
Figure 3.19 Kinetics of a non-competitive inhibitor.
The reaction pathway shows that the inhibitor binds both to free
enzyme and to enzyme complex. Consequently, Vmax cannot be
attained, even at high substrate concentrations.
Figure 3.20 Noncompetitive inhibition illustrated on
a double-reciprocal plot.
Irreversible inhibition
• Irreversible inhibitor tightly bound to the target
enzyme either covalently or noncovalently. Some
are important drugs.
• For example: penicillin can covalently modify the
glycopeptide transpeptidase, preventing the
synthesis of bacterial cell walls and thus killing
the bacteria. Aspirin can covalently modify
cyclooxygenase, reducing the synthesis of
inflammatory signals.
Active site
Figure 3.21 Formation of a penicilloyl-enzyme complex.
Penicillin reacts with transpeptidase to form an inactive complex,
which is indefinitely stable.
Figure 3.22 Schematic representation of the peptidoglycan in
Staphylococcus aureus. The sugars are shown in yellow, the
tetrapeptides in red, and the pentaglycine bridges in blue. The cell
wall is a single, enormous, bag-shaped macromolecule because of
extensive cross-linking.
Ⅶ. Regulation of enzyme activity
an organism is to coordinate its numerous
metabolic processes
1. Allosteric regulation:
allosteric enzyme regulated by molecules called
effectors (also modifiers)
no covalently binding at a site other than the active site
changing affinity of enzyme for its substrate
cooperativity– sigmoidal curve(V0 vs [S])
positive or negative effectors (feedback inhibition)
Effects of negative - or positive 
effectors on an allosteric enzyme.
A. Vmas is altered. B. The substrate
concentration that gives halfmaximal velocity(K0.5) is altered
2. Regulation of enzymes by covalent modification
• Phosphorylation and dephosphorylation
protein kinase, adenosine triphosphate (ATP)
phosphatases
• Response of enzyme to phosphorylation:
Phosphorylation may increase the activity (e.g.
glycogen phosphorylase) or decrease the activity
(e.g. glycogen synthase) of an enzyme
3. Induction and repression of enzyme synthesis
• Regulation of the amount of enzyme present
altering the rate of enzyme synthesis
• Slow regulation (hours to days)
• For example: elevated levels of insulin
cause an increase in the synthesis of key
enzymes involved in glucose metabolism
Ⅷ. Enzymes in clinical diagnosis
1. Plasma enzymes: secreted by certain cell
types (e.g. zymogen) and released from
cells during normal cell turnover
healthy persons – constant level – steady
state
Plasma is the fluid, noncellular part of
blood. Serum is obtained by centrifugation
of whole blood after its coagulation.
2. Alteration of plasma enzyme levels in disease
states
Tissue damage - release of enzymes into the plasma
Diseases: heart, liver, skeletal muscle and other
tissues
3. Plasma enzymes as diagnostic tools
Some enzymes in high activity in only one or a few
tissues: reflecting damage to the corresponding
tissue.
e.g. Alanine aminotransferase (ALT) is abundant in
liver. elevated level of ALT in plasma –hepatic
damage
4. Isoenzymes and diseases of the heart
Most isoenzymes (isozymes) catalyze the same
reaction with different primary structures.
Different organs contain different isoenzymes. The
pattern of isoenzymes found in plasma –the site of
tissue damage
e.g. Creatine kinase (CK) and lactate
dehydrogenase (LDH)
-myocardial infarction (when electrocardiogram
is difficult to interpret)
• Quaternary structure of isoenzymes:
containing different subunits in various
combinations.
e.g. CK, dimer, two kinds of subunits (M and B);
three isoenzymes: CK1=BB, CK2=MB,
CK3=MM (CK2 increased in myocardial
infarction)
• Diagnosis of myocardial infarction: myocardial
muscle is the only tissue that contains more than
5% of the total CK activity as the CK2 isoenzyme.
CK2 in plasma appears 4 to 8 hours following
onset of chest pain, and reaches a peak at 24
hours. LDH activity in plasma peaks 36 to 40
hours after infarction.
Figure 3.24 Subunit
structure and electrophoretic
mobility and enzyme
activity of creatine kinase
isoenzymes
LDH activity in
plasma peaks about
36 to 40 hours after
infarction
ACTIVITY
CK2 activity in
plasma peaks
about 24 hours
after infarction
Figure 3.25 Appearance of CK and LDH in
plasma after a myocardial infarction
Summary
Enzyme are protein catalysts that increase the
velocity of a chemical reaction by lowering the
energy of the transition state. Enzymes are not
consumed during the reaction they catalyze.
Enzyme molecules contain a special pocket or
cleft called the active site. The active site contains
amino acid side chains that create a 3-D surface
complementary to the substrate. The active site
binds the substrate, forming an ES complex. ES is
converted to enzyme-product (EP), which
subsequently dissociates to enzyme and product.
An enzyme allows a reaction to proceed rapidly
under conditions prevailing in the cell by providing
an alternate reaction pathway with a lower free
energy of activation. The enzyme does not change
the free energies of the reactants or products and,
therefore, does not change the equilibrium of the
reaction. Most enzymes show Michaelis-Menten
kinetics, and a plot of the initial reaction velocity,
V0, against substrate concentration, [S], has a
hyperbolic shape similar to the oxygen dissociation
curve of myoglobin. Any substance that can
diminish the velocity of such enzyme-catalyzed
reactions is called an inhibitor.
The two most commonly encountered types of inhibition
are competitive (which increases the apparent Km) and
noncompetitive (which decreases the Vmax). In contrast,
the multi-subunit allosteric enzymes frequently show a
sigmoidal curve similar in shape to the oxygen
dissociation curve of hemoglobin. They are frequently
found catalyzing the committed (rate-limiting) step of a
pathway. Allosteric enzymes are regulated by molecules
called effectors that bind noncovalently at a site other than
the active site. Effectors can be either positive or negative.
An allosteric effector can alter the affinity of the enzyme
for its substrate, or modify the maximal catalytic activity
of the enzyme, or both.