Protein Function and
Enzyme Kinetics
Manchuta Dangkulwanich, Ph.D.
General Biochemistry
Mahidol University International College
Week 3
T3 (2015-2016)
1
By the end of this section, you should be able to:
•
Describe the relations between enzyme catalysis of a reaction, the
thermodynamics of the reaction, and the formation of the transition
state.
•
Explain the relation between the transition state and the active site
of an enzyme, and list the characteristics of active sites.
•
Explain what reaction velocity is.
•
Explain how reaction velocity is determined and how reaction
velocities are used to characterized enzyme activity.
•
Identify the key properties of allosteric proteins, and describe the
structural basis for these properties.
•
List environmental factors that affect enzyme activity, and describe
how these factors exert their effects on enzymes.
2
Protein Function and Enzyme Kinetics
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Ligand-binding protein
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Association, Dissociation constant
•
Binding cooperativity, Hill plot
Enzymes - Type of enzymes
•
Cofactors, apoenzyme, holoenzyme
•
The mechanisms by which enzymes overcome the barrier
•
Enzyme Kinetics - The meaning of Vmax, KM, cat
•
Mode of reversible inhibition: competitive, noncompetitive, uncompetitive,
mixed
Regulation of enzyme activity
•
Allosteric, covalent modification, proteolytic cleavage
3
Proteins - classified by functions
•
Enzymes - catalytic activity and function
•
Transport Protein - bind & carry ligands
•
Storage Proteins - ovalbumin, gluten, casein, ferritin
•
Contractile (Motor): can contract, change shape, elements of cytoskeleton
(actin, myosin, tubulin)
•
Structural (Support): collagen of tendons & cartilage, elastin of ligaments
(tropoelastin), keratin of hair, feathers, & nails, fibroin of silk & webs.
•
Defense mechanism (Protect): antibodies (IgG), fibrinogen & thrombin, snake
venoms, bacterial toxins
•
Regulatory (Signal): regulate metabolic processes, hormones, transcription
factor & enhancers, growth factor proteins
•
Receptors (Detect stimuli): light & rhodopsin, membrane receptor proteins and
acetylcholine or insulin.
4
The functions of many proteins involve
the reversible binding of other molecules.
•
Ligand binds to a site on the
protein called the binding site.
•
Two models have been proposed:
Lock-and-key and Induced fit. The
model that is widely accepted in
the “Induced fit model”.
•
•
Lock-and-key model
The binding of a protein and ligand
is often coupled to a conformational
change in the protein: induced fit.
ligand
ligand-bound protein
Enzyme is a special class of
protein function. Enzymes bind and
chemically transform the
substrates in its catalytic or active
site.
protein
Induced fit model
http://molvisual.chem.ucsb.edu/ABLE/induced_fit/index.html
5
Reversible binding of a protein to a ligand:
Oxygen binding protein
Model Protein: Myoglobin
Porphyrin
Heme: Protoporphyrin IX + Fe2+
6
•
The heme group is
present in myoglobin,
hemoglobin, and many
other proteins. Heme
consists of a
protoporphyrin IX and
Fe2+.
•
Iron atom of heme has
six coordination. Four of
which are bonded to
the protophyrin plane.
One binds to His
residue in the protein.
Protein-ligand interactions can be described quantitatively.
P + L ⇌ PL
•
Ka =
•
𝛳=
•
[PL]
[P][L]
association constant
binding sites occupied
total binding sites
[PL]
[L]
=
=
[PL] + [P]
[L] + 1/Ka
Ka = 1/Kd
•
7
dissociation constant
Graphical representations of ligand binding
•
Kd is the concentrations of ligands where half of the proteins are bound.
•
Kd is often used to expressed the affinity of enzymes. A lower values of Kd
corresponds to a higher affinity of ligand to the protein.
•
In the right figure, which version of the protein (X or Y) bind tighter to Ligand A?
8
9
Hemoglobin undergoes a structural change on binding oxygen
PDB ID: 1HGA
PDB ID: 1BBB
•
Oxygen has a significantly higher affinity for hemoglobin in the R
state. When oxygen is absent, the T state is more stable and is
thus the predominant conformation of the deoxyhemoglobin.
•
Binding of oxygen to the T state triggers a change to the R
state.
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Enzyme - Biological catalyst
•
In enzymatic reactions, the molecules at the beginning of the process
are called substrates, and the enzyme converts them into different
molecules or the products.
•
Enzymes are not consumed or destroyed after the reactions.
•
How do enzymes effect the kinetics and thermodynamics of reactions?
11
Enzymes are classified on the basis of
the types of reactions that they catalyze
Class
Type of reaction
Example
1. Oxidoreductaces
Oxidation-reduction
Lactate dehydrogenase
2. Transferases
Group transfer
Nucleoside monophosphate
kinase (NMP kinase)
3. Hydrolases
Hydrolysis reactions (transfer of
functional groups to water)
Chymotrypsin
4. Lyases
Addition or removal of groups to form
double bonds
Fumarase
5. Isomerases
Isomerization (intramolecular group
transfer)
Triose phosphate isomerase
6. Ligases
Ligation of two substrates at the
expense of ATP hydrolysis
Aminoacyl-tRNA synthetase
12
Enzymes affects the reaction rates, not the equilibria
•
To describe the free-energy changes for reactions, chemists define a standard set of
conditions (temperature 298 K; partial pressure of each gas 1 atm; concentration of
each solute 1 M ) and express the free-energy change for this reacting system as
∆Gº, the standard
free-energy change. Because biochemical systems commonly
+
involve H concentrations far below 1 M, biochemists define a biochemical standard
free-energy change, ∆G’º , the standard free-energy change at pH 7.0.
13
Enzymes accelerate the reaction rate
•
Enzymes accelerate the
attainment of equilibria but do
not shift their positions. The
equilibrium is a function of
only the free-energy different
between reactants and
products.
•
The same equilibrium is
reached (the same amount of
product).
•
However, this same
equilibrium point is reached
much more quickly in the
presence of an enzyme.
14
Many enzymes require cofactors for activity
•
Apoenzyme + cofactor = holoenzyme
•
Cofactors can be subdivided into metal
ions and organic molecules, called
coenzymes. Often derived from vitamins,
coenzymes can be either tightly or loosely
bound to the enzyme. If tightly bound,
they are called prosthetic groups.
15
Enzyme enhances reaction rates by 5 to 17 order of magnitude
•
Forming many weak bonds
and interactions between an
enzyme and its substrate in
ES complex releases free
energy, called binding
energy, which contributes to
specificity as well as to
catalysis.
16
The active sites of enzymes have some common features
•
The active site of an enzyme is the
region that binds the substrates. It
contains the amino acid residues
that directly participate in the
making and breaking of bonds.
•
The interaction of the enzyme and
substrate at the active site promotes
the formation of the transition state.
•
The active site is a three-dimensional cleft or crevice formed by groups that come
from different parts of the amino acid sequence.
•
The active site takes up a small part part of the total volume of an enzyme.
•
Active sites are unique microenvironments.
•
Substrates are bound to enzymes by multiple weak attractions.
•
The specificity of binding depends on the precisely defined arrangements of atoms
in an active site.
17
Weak interactions are optimized in the transition state
•
•
•
•
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ES is more stabilized than S.
TS has high energy.
Increased activation energy!!!
TS has low energy.
Reduced activation energy!!!
‡
Binding energy is used to overcome the ΔG , energy barrier
•
A reduction in entropy, in the form of
decreased freedom of motion of two
molecules in solution
•
The distortion of substrates upon
binding to the enzymes is supported by
binding energy.
•
The need of proper alignment of
catalytic functional groups on the
enzyme.
•
Induced fit mechanism proposed
by Daniel Koshland in 1958.
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Conformational change of the
enzyme to bring specific
functional groups on the enzyme
into the proper position to
catalyze the reaction.
19
Specific catalytic groups contribute to catalysis
1. General acid-base catalysis
2. Covalent catalysis
3. Metal ion catalysis
20
Enzyme Kinetics
An approach to understanding mechanism
E + S ⇌ ES ⟶ E + P
d[P]
Rate =
dt
21
Enzyme Kinetics
An approach to understanding mechanism
E + S ⇌ ES ⟶ E + P
d[P]
Rate =
= slope
dt
•
•
•
Initial rate or initial velocity (V0) when [S] >> [E]
[E] is usually in nM while [S] is five or six orders of
magnitude higher.
In the beginning of the reaction [S] is regarded as
constant.
22
Michaelis-Menten Kinetics
1. Fast reversible combination of E & S
k1
E + S ⇌ ES
k-1
2. ES Breaks down in a slower step, rate-limiting
k2
ES ⟶ E + P
•
•
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Steady state approximation: the concentrations of
intermediates, [ES], remain constant over time.
Vmax [S]
Michaelis-Menten equation: V0 =
KM +[S]
Vmax = k2[E]0 and KM = (k2 + k-1)/k1
23
Dependence of initial velocity on substrate concentration
Vmax [S]
V0 =
KM +[S]
[S] = KM
V0 = Vmax /2
[S] >> KM
V0 = Vmax
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[S] << KM
Vmax [S]
V0 =
KM
The double reciprocal plot, or Lineweaver-Burk plot
•
The double reciprocal
plot is another mean to
determine KM and Vmax.
•
Algebraic manipulation
of the basis MichaelisLenten equation to
gives a straight line plot.
Vmax [S]
V0 =
KM +[S]
1
KM
1
=
+
V0 Vmax [S] Vmax
25
Interpreting Vmax and KM
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The KM values of enzymes range widely. For most enzymes, the KM lies
between 10-1 and 10-7 M
•
The KM is equal to the concentration of substrate at which half of the active
sites are filled. Thus, KM provides a measure of the substrate concentration
required for significant catalysis to take place.
•
The KM value provides an approximation of substrate concentration in vivo,
which in turn suggests that most enzymes evolved to have significant activity at
the substrate concentration commonly available.
26
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For 2-step mechanism, KM = (k2 + k-1)/k1
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When k2 << k-1, KM = k-1/k1
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KM = Kd, dissociation constant, = k-1/k1
Vmax
•
The maximal velocity Vmax reveals the turnover number of an enzyme,
which is the number of substrate molecules that an enzyme can convert
into product per unit time when the enzyme is filled saturated with
substrate.
•
The turnover number is equal to the rate constant k2, which is also called
kcat. If the total concentration of active sites [E]T is known, then:
Vmax = k2[E]T
•
•
and
k2 = Vmax /[E]T
-6
For example, a 10 M solution of carbonic anhydrase (1 active site per
subunit) catalyzes the formation of 0.4 M HCO3- per second when the
5 -1
enzyme is fully saturated with substrate. Hence, k2 is 4×10 s .
The turnover number of most
enzymes with their physiological
substrates fall in the range from 1
to 104 per second.
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kcat and KM signifies the kinetic efficiency of enzymes
•
Specificity constant, kcat/KM, is used to compare the catalytic efficiencies
of different enzymes or the turnover of different substrates. It takes into
account both the rate of catalysis with a particular substrate (kcat) and the
nature of the enzyme-substrate interaction (KM).
•
Upper limit of the kcat/KM is diffusion controlled in the range of 10 to 10
-1 -1
M s .
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9
Enzymes are subject to reversible or irreversible inhibition
•
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Inhibitors of enzymes are
among the most important
pharmaceutical agents.
•
HIV protease with a bound
inhibitor, darunavir, at the
interface of the dimer.
Three kinds of reversible inhibition
1. Competitive inhibition
3. Non-competitive inhibition
2. Uncompetitive inhibition
29
Reversible inhibitors
Substrate binds to an enzyme’s active site
to form an enzyme-substrate complex.
A competitive inhibitor binds at the active site
and thus prevents the substrate from binding
An uncompetitive inhibitor binds only to the
enzyme substrate complex.
A noncompetitive inhibitor does not prevent
the substrate from binding.
30
1. Competitive Inhibition
It works by reducing the proportion of enzyme molecules bound to a substrate.
where [I] is the concentration of inhibitor and KI is the
dissociation constant for the enzyme-inhibitor complex.
As the value of [I] increases, the value of KMapp
increases. In the presence of a competitive inhibitor, an
enzyme will have the same Vmax as in the absence of
an inhibitor.
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2. Uncompetitive Inhibition
It works by binding to the ES complex and ESI does not result in product.
Vmax
V0
Vmax'
KM’ KM
•
[S]
At high concentrations of substrate, V0
approaches Vmax/𝛼’. Thus, an uncompetitive
inhibitor lowers the measured Vmax. Apparent KM
also decreases, because the [S] required to
reach one-half Vmax decreases by the factor 𝛼’.
32
3. Noncompetitive Inhibition
A substrate can bind to both E and EI and ESI cannot form product.
S
E
kcat
ES
I
Ks
Ki
EI
Kss
Kii
E + P
I
EIS
S
•
The inhibitor binds to both the free enzyme and the
ES complex with the same affinity. Vmax cannot be
attained even at high substrate concentrations. KM
will remain the same.
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Enzyme inhibition mechanism
34
Enzyme inhibition plots
35
Effect of reversible inhibitors on
apparent Vmax and apparent KM
Inhibitor type
Apparent Vmax
Apparent KM
None
Vmax
KM
Competitive
Vmax
αKM
Uncompetitive
Vmax/α’
KM/α’
None competitive
Vmax/α’
KM
36
Irreversible Inhibitors
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binds covalently with or destroy the functional groups on an enzyme that is
essential to the activity
•
or forms a particularly stable noncovalent association
Bromoacetol phosphate, an analog of
dihydroxyacetone phosphate, binds at the active
site of the enzyme and covalently modifies a
glutamic acid residue required for enzyme activity.
Enzyme Inhibition by Diisopropylphosphofluoridate
(DIPF), a Group-Specific Reagent. DIPF can inhibit an
enzyme by covalently modifying a crucial serine residue
37
Suicide Inactivator or mechanism based inhibitors
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A unique class of irreversible inhibitor that is important in rational based drug
design
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It undergoes the first few chemical steps of the normal enzymatic reaction, and
combines irreversibly with the enzyme.
Monoamine oxidase, an enzyme
important for neurotransmitter
synthesis, requires the cofactor
FAD (flavin adenine dinucleotide).
N,N-Dimethylpropargylamine
inhibits monoamine oxidase by
covalently modifying the flavin
prosthetic group only after the
inhibitor is first oxidized. The N-5
flavin adduct is stabilized by the
addition of a proton.
38
Regulation of enzyme activity
Groups of enzymes work together in sequential pathways to
carry out a given metabolic process. A common mean of
biochemical regulation is feedback inhibition.
The presence of the product inhibits the enzyme that
catalyzes the committed step of the pathway.
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Three ways to regulate enzyme activity
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Allosteric controls
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Covalent modification
•
Proteolytic cleavage
40
Allosteric regulation
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Allosteric enzymes function through reversible, noncovalent binding
of regulatory compounds called allosteric modulators or allosteric
effectors, which are generally small metabolites or cofactors.
•
Allosteric regulators bind to a distinct allosteric site, NOT the active
site, and alter the shape of the active site.
41
Structural changes in a multi-subunit protein
under going cooperative binding to ligand
•
An allosteric protein is one in
which the binding of a ligand
to one site affects the binding
properties of another site on
the same proteins.
•
When the modulator are
identical, the interaction is
termed homotropic. When the
modulator is different from the
normal ligand, the interaction
is heterotrophic.
42
Aspartate transcabamoylase (ATCase) catalyzes the first
committed step in the pyrimidine biosynthetic pathway
•
CTP and UTP, the two end products of the pyrimidine
biosynthesis, are the negative allosteric effectors of ATCase.
Their actions prevent the pyrimidine surplus.
•
The effectors inhibit ATCase activity, establishing a negative
feedback loop.
43
Allosteric enzyme exhibit a sigmoid kinetic behavior
•
Rather than the hyperbolic curve of
V0 and [S] in typical MichaelisMenten kinetics, allosteric
enzymes show a sigmoidal
saturation (S) curve.
•
Allosteric enzymes transition from a less active state to a more active state
within a narrow range of substrate.
•
The activity of allosteric enzymes is more sensitive to changes concentrations
near KM than are Michael-Lenten enzymes with the same Vmax.
44
Covalent modification
Regulation of glycogen phosphorylase activity
by covalent modification. In the more active form
of the enzyme, phosphorylase a, specific Ser
residues, one on each subunit, are phosphorylated
by phosphorylase kinase. Phosphorylase a is
converted to the less active phosphorylase b by
enzymatic loss of these phosphoryl groups,
promoted by phosphorylase phosphatase.
45
Some enzymes and other proteins are regulated by
proteolytic cleavage of an enzyme precursor
•
Zymogen is an inactive precursor of proteases that is converted to an active form by an
action of another enzyme. Specific cleavage causes conformational changes that
expose the enzyme active site.
•
Other enzymes are more generally called proproteins or proenzymes. For example, the
protein collagen is synthesized as the soluble precursor procollagen.
46