Transcript Chap 7. Detection of Intermediates in Enzymatic Reactions

Chap 12. Enzyme-Substrate
Complementary and the Use of
Binding Energy in Catalysis
• Catalysis in model systems is still many orders of
magnitude short of that found in enzymes
• Enzymes have evolved to use the binding energy between
enzymes and substrates to provide the additional catalysis
• The binding energy can be used to lower chemical
activation energies
• The use of binding energy lowers the activation energy of
kcat/KM and the activation energy of kcat
A. Utilization of Enzyme-Substrate
Binding Energy in Catalysis
1. Binding Energy Lowers the Activation Energy
of kcat/KM
E+S
E+S
ΔGT‡
KM
ΔGS
ES
kcat/KM
ΔGT‡
kcat
ΔG‡
products
ES‡
= ΔG‡
+ ΔGS
kcat
kT
RT ln
 RT ln
 G ‡  GS
KM
h
• ΔGT‡ is activation energy proportional to kcat/KM (positive)
• ΔG‡ due to bond breaking/making (positive)
• ΔGS is binding energy of the substrate (negative)
2. Interconversion of Binding and Chemical
Activation Energies
• Complementary structure: the maximum binding energy
• Complementary to the structure of the TS state
 lowering the activation E of kcat
• Complementary to the structure of the unaltered substrate
 increasing the activation E of kcat
[S] > KM ( = kcat[E]0)
• Stabilizing both ES and TS:
no catalytic advantage
• Stabilizing ES only:
increasing the activation E of
kcat
and decreasing the reaction rate
• Stabilizing TS only:
lowering the activation E of
kcat
and increasing the reaction rate
[S] < KM (v = kcat/KM[E]0[S])
• Stabilizing both ES and TS:
lowering the activation E
and increasing the reaction rate
• Stabilizing ES only:
no catalytic advantage
• Stabilizing TS only:
lowering the activation E of kcat
and increasing the reaction rate
B. Experimental Evidence for the Utilization of
Binding Energy in Catalysis and Enzyme-TS
Complementarity
• for chymotrypsin and elastase,
larger leaving groups increase kcat/KM (effect is almost
all kcat)
• for pepsin
larger side chain groups or additional amino acids raises
kcat, while KM stays at ~0.1mM
C. Evolution of the Maximum Rate: Strong
Binding of the Transition State add Weak
Binding of the Substrate
• Better binding of TS than the substrates: maximizing kcat/KM
• The maximum reaction rate for a particular concentration
depending on the individual kcat and KM
KM < [S]
KM > [S]
weak binding
High KM Gives a Lower Activation Energy
KM < [S]
- low KM leads to a
thermodynamic “pit”
KM > [S]
- high KM leads
to “a step up the
thermodynamic ladder”
If KM >> [S], [E]0 = [E]
kcat
v  [ E ][S ]
KM
constant kcat/KM and [S]
• KM = [S], half unbound of the enzyme = 50% of the maximum
• KM = 5[S], 5/6 unbound of the enzyme = 83% of the maximum
Control Enzymes are Exceptions to the Principle
of High KM’s
• Control enzymes: evolved for the purpose of regulation
• The fist enzyme on metabolic pathway:
A low KM may be advantage
• Hxokinase: the first enzyme in glycolysis
KM = 0.1 mM, [glucose] = 5 mM
The KM/[S] Values of Most Enzymes in Glycolysis
are in the range of 1 to 10 and 10 to 100
The glycolytic enzymes
The majority of the enzymes
are in the 1< KM/[S] < 10 range
Regulatory enzymes would
likely be here
(e.g., hexokinase)
The Perfectly Evolved Enzyme for Maximum Rate
have Maximum kcat/KM and High KM
• kcat/KM = 108 to 109 s-1 M-1
• KM > [S]
• Ex. carbonic anhydrase and trisephosphate isomerase:
D. Molecular Mechanisms for the Utilization of
Binding Energy
1. Strain:
substrates distorted to make the transition state contact better
with the enzyme
2. Induced fit:
the enzyme distorted after binding occur
3. Nonproductive binding:
not a mechanism for increasing KM,
but has a qualitatively similar effect on enzyme catalytic rate
Induced Fit Requires the Energy to Distort Enzyme
Eact
K
Ein
KM
S
K’M
S
EactS
K’
EinS
kcat
K << 1
(kcat/KM)obs = K(kcat/KM)
K’ >> 1
(kcat)obs = kcat
(KM)obs = KM/K
• If all enzymes are in the active conformation,
kcat is unchanged and KM is higher
Thus, slows down catalysis (kcat/KM)
• Importance of induced fit:
providing the means of access of substrates when the TS
needs to be completely surrounded by groups on the enzyme
Strain, Induced Fit, and Nonproductive Binding
do not Alter the Specificity
• Altering kcat and KM in a mutually compensating manner
without changing kcat/KM