1. 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

2. A. Utilization of Enzyme-Substrate Binding Energy in Catalysis
1. Binding Energy Lowers the Activation Energy of kcat/KM
E + S ES products KM
kcat
ΔGS
ΔG‡
E + S ES‡
kcat/KM
ΔGT‡
ΔGT‡ = ΔG‡ + ΔGS
ΔGT‡ is activation energy proportional to kcat/KM (positive)
ΔG‡ due to bond breaking/making (positive)
ΔGS is binding energy of the substrate (negative)

3. 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

4. [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

5. [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

6. 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

7. 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

8. 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]
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

9. 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

10. 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)

11. 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:

12. D. Molecular Mechanisms for the Utilization of Binding Energy
Strain:
substrates distorted to make the transition state contact better
with the enzyme
Induced fit:
the enzyme distorted after binding occur
Nonproductive binding:
not a mechanism for increasing KM,
but has a qualitatively similar effect on enzyme catalytic rate

13. Induced Fit Requires the Energy to Distort Enzyme
K << 1
(kcat/KM)obs = K(kcat/KM)
K’ >> 1
(kcat)obs = kcat
(KM)obs = KM/K KM
kcat
Eact EactS S K K’
K’M
Ein EinS S
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

14. Strain, Induced Fit, and Nonproductive Binding do not Alter the Specificity
Altering kcat and KM in a mutually compensating manner without changing kcat/KM