Chemical Biology I TIGP0101-00 Enzyme Kinetics B807 TEL:27898662 Institute of Chemistry Academia...
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Transcript of Chemical Biology I TIGP0101-00 Enzyme Kinetics B807 TEL:27898662 Institute of Chemistry Academia...
Chemical Biology ITIGP0101-00
Enzyme Kinetics B807 TEL:27898662 Institute of Chemistry
Academia Sinica Fall, 2003
Mechanisms: A P1. Kinetics: steady state/pre steady state2. Spectroscopic: structure(or active site) NMR/EPR (fluorescence)3. X-ray: structure determination at active site4. Binding studies: thermodynamic understanding inhibitors transition states. sequence analysis, genomics, genetic manipulation DNA protein sequence mutants
Enzymes: A rate P
T.S.
A
P
overcomeenergy barrier: (1). lower barrier stabilize T.S. (2). destabilize ground state or enzymes substrates (A)
1. rate acceleration: how fast?2. specificity: how selective?
destabilize ground state
or enzymes substrates (A)
lower barrier stabilize T.S.
Rate acceleration: non-enzyme very slow How fast of reaction rate enzyme will facilitate?
*turnover number: the number of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is saturated with substrate.
Vmax moles/min Et moles enz. min-1
1 106, s-1
usually average’s rate, 6 s-1
EX1: UreaseEX2: CatalaseEX3: Carbonic anhydrase
Catalytic Power
Specificity: Enzymes’ active envolved to do some specific things.EX1: Hexokinase phosphorylation:
O
OH
OH
OH
HO
OH + ATPO
OH
OH
OH
O
OH
P
O
O
O
+ ADP 106
rel rate
HO H + ATP OP
OH
HO
O+ ADP 1
:
O
OH
OH
OH
OH C-6 is missing 103
:
EX2: Alcohol dehydrogenase:
Enzyme commission Systematic Namenumber
1 Oxidoreductases(oxidation-reduction reactions)2 Transferases(transfer of functional groups)3 Hydrolases(hydrolysis reaction)4 Lyases(addition to double bonds)5 Isomerases(isomerization reactions)6 Ligases(formation of bonds with ATP cleavage)
Example: EC 1.1.1.1 alcohol dehydrogenase EC 2.1.1.1 nicotinamide N-methyltransferase EC 3.3.1.21 -glucosidase EC 4.1.1.1 pyruvate decarboxylase EC 5.3.1.1 triose-phosphate isomerase EC 6.5.1.3 RNA ligase
Steady state: A P
x
x
x
x
x
x x x x xrate
[A]
first order on [A]
mixed order
zero-orderon [A]
E EA P + E
K1A
K2
K3
EP
Steady-state assumption: 1925, G. E. Briggs and James B. S. Haldance assuming the concentration of the enzyme-substrate complex(EA) quickly reaches a constant value in such a dynamic system.
That is, EA is formed as rapidly from E + A as it disappears byits two possible fates: dissociation to regenerate E + A, and reaction to form E + P.
d[EA] dt = 0 d[E]
dt = 0
A + B P + Q
Nomenclature: by Clelandsubstrates A, B, C, D,.....etcproducts P, Q, R, S,......etcinhibitors I, J, K,......etcenzyme complex E, F, G(stable complex) enzyme complex EA(unstable transitory complex)
enzyme complex EAB EPQ(central complex)
E : free enzymeF : covalent attachementenzyme complex
x
x
x
x
x
x x x x xrate
[A]
first order on [A]
E EA P + E
K1A
K2
K3
EP
Steady state: Michaelis Menten equation =
VmaxAKa + A
reciprocal 1 =
VmaxAKa + A
= +Ka
VmaxA Vmax
1
Derivation of Rate Equations (Biochemistry, 1975, 14, 3320)
1Ka
1/
1/A
1/V
slopek/V
E EA P + E
K1A
K2
K3
EP
rate = dP dt = k3 [EA]
rate = dP dt = k3 [EA]
d[EA] dt = [E]K1A K2[EA] K3[EA]
d[E] dt = K3[EA] K2[EA] K1A[E]
ET = E EA Steady state assumption:
d[EA] dt = 0 d[E]
dt = 0Solve for EA
E = ET EA K1A(ET EA) K2(EA) K3(EA) = 0 K1AET K1A(EA) K2(EA) K3(EA) = 0K1AET = EA(K1A K2 K3)
EA = (K1A K2 K3)
K1AET
because
rate = dP dt = k3 [EA] = k3
(K1A K2 K3)
K1AET
E EA P + E
K1A
K2
K3
EP
rate = dP dt = k3 [EA] = k3
(K1A K2 K3)
K1AET
rate
ET
= (K1A K2 K3)
k3K1A divide by K1
= VmaxAKa + A
= k3A
A K1
K2 K3
(1) rate as A , k3 is predominate, k3 = Vmax
(2) rate as A 0, K1A 0 ,
rate
ET
= (K2 K3)
k3K1A
(K2 K3)
k3K1 = V
Kbecause K3 = Vmax
K = K1
(K2 K3)
(initial rate)
A P
E EA P + E
K1A
K2
K3EP
K4
K5
Steady-state Rate Law for a One-substrate,One-product Reaction with Two Reversible Steps
binding chemical dissociation
Replacing every equilibrium rate constant by net rate constant:Net rate constant:
E1 E2 E3 E1
K1 K3
K5
steady state
each [E] depend on next net rate constant K magnitudeif K3
large, [E2]
if K3 small, [E2]
Therefore, E1 K1
1,
E1
Et
=K1
1
K1
1
K3
1
K5
1+ +
•Flux is constant at steady state:
rate =E1(K1) =E2(K3
) =E3(K5) at steady state
velocity = E1(K1) =
ET
{because }E1
Et
=K1
1
K1
1
K3
1
K5
1+ +
K1
1
K3
1
K5
1+ +
Et
= 1
n
i Ki
1
<Homework> Go back to derive an equation for a one-substrate,one-product reaction with one reversible steps
1 =
VmaxAKa + A
= +Ka
VmaxA Vmax
1
Lineweaver-Burk double-reciprocal plot
Kinetic Mechanisms
forward V1 ; reverse V2
Michaelis complexes Ka, Kb
inhibition constants(thermodynamic)Kia, Kib
(A). Sequential mechanism: All substrates bind before chemical events. 1. Order: Enzyme binds in different order with substrates. If the mechanism is ordered, the substrates will add to the enzyme as A first, B second, etc., and the first product to dissociate from the enzyme will be P, followed by Q etc. (a). Order sequential mechanism: NAD+-dependent dehydrogenases
E EA
K1A
K2
K3B
K4
EABK5
K6
EPQK7
K8P
EQ EK10
P
K9
E EA EAB
Order sequential mechanism:
A B P Q
E EA (EAB EAP) EQ E
(b). Theorell-Chance mechanism:steady state concentration of central complexs are low.
A B P Q
E EA EA E
*It may be impossible for B to bind until after A binds and promotes aconformational change in the enzyme that exposes the B binding site.
example: liver alcohol dehydrogenase.
2. Random:A enzyme catalyzing a random mechanism would possess two distinct sites, one for each substrate(orproduct), so that the reaction of one substrate with the enzyme may occur before or after the other.
E
EA
EBEAB
A B P Q
B A Q P
E EEAB
EPQ
(a). Ordinary random mechanism: if slowest step is one other than the interconversion of the central complex, EAB EPQ. (no enzyme is known to have this mech.)(b). Random-rapid equilibrium mechanism: If the slowest is central complex. example:yeast hexokinase, creatine kinase.
(B). Ping Pong mechanism: Chemistry occurs prior to binding of all substrates
The addition of one substrate to the enzyme causes a reactionwhich results in the formation of one product and a new stableform of the enzyme which in turn reacts with the second substrates. examples: thioltransferase, phosphoglucomutasetransaminase.
A P B Q
E (EA FP) F FB EQ E
a new stable form of the enzyme
Kinetics of Enzyme-catalyzed Reactions Involving Two or more Vary Substrates
A + B P + Q
1. Intersecting Pattern:indicates sequential combination of both substrates prior to release of a product.
1/
1/A
[B]
= V1AB
KiaKb + KaB + KbA + AB
1/
1/B
[A]
Kinetics of Enzyme-catalyzed Reactions Involving Two or more Vary Substrates
A + B P + Q
2. Parallel Pattern: An irreversible step intervenes between the timesof combination of the two substrates in the mechanism.
1/
1/A
[B]1/
1/B
[A]
= VAB
KaB + KbA + AB
Kinetics of Enzyme-catalyzed Reactions Involving Two or more Vary Substrates
A + B P + Q
3. Equilibrium Ordered Pattern:
= VAB
KiaKb + KbA + AB
Since it corresponds to ordered addition of A and B, with addition of A at equilibrium, looks different when [A]and [B] are varied.
1/
1/B
[A]1/
1/A
[B]
•This pattern is most commonly seen with metal activators which are not consumed during the reaction, but must be present to permit substrate binding.
Slope and Intercept
intercept---velocity at sat. substrate , observe intercept. A BSlope---rate at low substrate concentration
A B P Q
E EA (EAB EAP) EQ E
*Sequential mech.
intercept change enzyme different
A EA B EABE EA
slope will change if change [B]
A P B Q
E (EA FP) F FB EQ E
Slope and Intercept
*Ping pong mech.
intercept changeslope no slope effect by change [B]
Enzyme Inhibition
product, dead-end substrate inhibited enzyme
1. Competitive inhibition (C): A competitive inhibitor is a substance that combines with free enzyme in a manner that prevents substrate binding. That’s, theinhibitor and the substrate are mutually exclusive, often becauseof true competition for the same site.
1/
1/A
[I]
Slope change onlyVmax is the same
Competitive inhibition (C):
Active siteof enzyme
Substrate
Inhibitor
Products
Inhibitor preventsbinding of substrate
Substrate and inhibitorcan bind to the active site
Enzyme Inhibition
2. Uncompetitive inhibition (UC):A classical UC inhibitor is a compound that binds reversibly to the enzyme-substrate complex yielding an inactive ESI complex. The I does not bind to free enzyme.
1/
1/A
[I]
E + A EA P + E
+
I
EAI NO REACTION
KI
K1
K2
K3
Intercept changeSlope is the same
Enzyme Inhibition3. Noncompetitive inhibition (NC):
A classical NC inhibitor has no effect on substrate bindingand vice versa, A and I bind reversibly, randomly and independently at different sites.
1/
1/A
[I]
Slope changeIntercept change
Noncompetitive inhibition (NC):
Active site
Inhibitor site
Binding ofinhibitordistorts theenzyme
In the absenceof inhibitor,products areformed
Substrate andinhibitor can bindsimultaneously
The presence ofthe inhibitorslows the rate ofproduct formation
Effects of Inhibitors on Michaelis-Menten Reactions
Type ofInhibition
Michaelis-Menten Equation
Lineweaver-Burk Equation
Effect of Inhibitor
NoneVmaxA
Km + A =
1
= + Km
VmaxA Vmax
1
None
CompetitiveVmaxA
Km + A =
1
= +Km
VmaxA Vmax
1
Increase Km
UncompetitiveVmaxA
Km+ ’A =
1= +
Km
VmaxA Vmax
’ Decrease Km and Vmax
NoncompetitiveVmaxA
Km+ ’A =
1= +
Km
VmaxA Vmax
’Decrease Vmax; may increase or decrease Km
= 1 + [I]/KI ’ = 1 + [I]/K'I
1/
1/A
[I]
Intercept Idea:competitive pattern
if A No inhibition by [I]
1/A 0
I and A competiting for the same site(for the same enzyme)No intercept
I and A bind to different enzyme intercept effectwill become NC inhibition
Exceptions:
Slope effect:
EAK1A
K2
raised
E
E EA respect
lower EEA respect
I reversibly connected to either or EA E
show slope effectactual product inhibitors
example: dead-end inhibitor
Catalysis
1. Covalent catalysis:rate acceleration from the formation ofcovalent bonds between enzyme and substrate.
Enz-X: better attacking group and better leaving groupexample: ping-pong mechanism
smaller
2. Acid/base catalysis:(a) specific acid-base catalysis(b) general acid-base catalysis
N N
O
H3CHN NO
H3C
H2O
HO H
N NH
O
+
This reaction accelerated by imidazole.Usually increasing concentration ofproduct(imidazole) will decrease the rate.However, imidazole help to extract H+fromwater molecules in T.S.
general acid-base catalysis
3. Entropy: entropy loss in the formation of EA
The rotational and translational entropies of the substrate have been lost already during formation of EA complex
example: Strain/distortion
Transition state:Enzyme stablize T.S. to accelerate the reaction rate.Enzyme should bind tighter in T.S. than in substrate and product states.
example: Proline racemase and Isocitrate lyase (Prof. Robert Abeles)