1. Time to Connect……. Enzyme Activity is amount of enzyme that
converts one micromole of substrate
to product in one minute
Velocity is micromoles product
formed per minute
This slide is intended to show the connection between enzyme activity and
Vmax.
Vmax is maximum product formed
at saturating substrate per minute
for a given amount of enzyme,

2. Km meaures the affinity of the enzyme for the
E + S [ES] E + P
There are 2 steps in an enzyme-catalyzed reaction
1. Formation of ES Km
2. Formation of P
kcat
Km meaures the affinity of the enzyme for the
substrate. It does not concern product formation
Kcat measures how rapidly the product is formed
from ES. It does not measure binding of substrate

3. Time to Connect……. Vmax = kcat [E] Kcat = k2 when ES = ET or Vmax
Therefore:
Vmax = kcat [E] or
Kcat =
Vmax
[E]
mmoles S per minute
mmoles Enzyme =
Turnover
kcat is synonymous with Turnover Number

4. Multiple the numerator by the reciprocal of the denominator
Time to Get Efficient…….
At maximum efficiency, k1 controls the rate k2
k2 + k-1 k1
kcat / Km =
Multiple the numerator by the reciprocal of the denominator
Every collision results in ES
k1k2
k2 + k-1 = k2
When an enzyme is maximally efficient, k2 >>k-1 k1 =
kcat / Km of 108 or 109 means
diffusion control

5. kcat kcat/Km Km(M) Chymotrypsin 0.14 9.3 Urease 0.025 1 x 104 4 x 105
(Per second)
Chymotrypsin
0.015
0.14
9.3
Urease
0.025
1 x 104
4 x 105
Acetylcholine esterase
1.4 x 104
1.5 x 108
Catalase
0.025
1 x 107
4 x 108
The rate of catalysis by Acetylcholine esterase and catalase is controlled by diffusion …
Every collision with the enzyme results in a product

6. Metal Ions in Catalysis-
One third of all enzymes require a metal ion for catalysis

7. Zn 2+Polarizes H2O, making it a better nucleophile
His –Zn2+
His O H ..
+ C
His –Zn2+
His O H C
H2O
His –Zn2+
His O H ..
+ H+
+ H C
Bicarbonate
Displaces HCO3-

8. RNA Hydrolysis . .. O O-P-O-CH2 Base O-P H CH2 O O-P-O-CH2 Base H N P+ ..
2’,3’-cylic
nucleotide N .
H2O
His 12 O
Base
O-P
CH2 H O H N H +
His 119

9. RNA Hydrolysis (cont.) : = 3’ end of split RNA O O-P-O-CH2 Base O O N
His 12 O P = O O H
3’ end of split RNA H N +
His 119 + N

10. Insights into the Mechanism of a Digestive
Enzyme
1. Some digestive enzymes catalyze the
hydrolysis of proteins. Examples are:
trypsin and chymotrypsin
2. Some have a Serine residue at the catalytic site
3. Histidine and Aspartate are also needed
4. The 3 make up a catalytic triad

11. Catalytic Triad of Chymotrypsin:
Histidine C O
CH2 CH HN
O=C : H O
CH2 C= N CH ..
Attacking Group
on Serine
Aspartate
Catalytic
Site
Serine

12. Chymotrypsin Hydrophobic Pocket O C Asp N H His Ser .. HO—C — CH—
NH—C —CH —NH —A-A-A-A-A-A-A-A- O R
Hydrophobic Pocket

13. Chymotrypsin Split Protein O C Asp N H His Ser C —CH —NH —A-A-A-A-A-A-
AA—C —
CH—NH3 O R

14. Chymotrypsin Split Protein O C Asp N H His Ser H O :
C —CH —NH —A-A-A-A-A-A
Split
Protein O
AA—C —
CH—NH3 O R

15. Chymotrypsin Broken Peptide Bond O C Asp N H His Ser H
C —CH —NH —A-A-A-A-A-A O O
AA—C —
CH—NH3 O R

16. -chymotrypsin (active) -chymotrypsin (active) Zymogens
Rule: Many hydrolytic enzymes are synthesized as inactive precursors. The suffix “ogen”, or the prefix pro, and prepro” designate a precursor
Rule: Activation requires removal of a blocking peptide by proteolytic cleavage
Trypsin
COOH
S - S
Chymotrypsinogen
(inactive)
COOH
S - S
-chymotrypsin
(active)
Chymotrypsin
COOH
S - S
-chymotrypsin
(active)

17. Hexokinase Random More than One Substrate….. E + S1 + S2
E + S1 then S2
E-S1-S2
E + P1 + P2
E + S2 then S1
Hexokinase
Example
Glucose + ATP
E + Glucose
E-Glucose
+ ATP or
E-Glucose-ATP
E + ATP
E-ATP
+ Glucose

18. ORDERED More than One Substrate….. E + S1 + S2 E + S1 E-S1 E-S1-S2
No Reaction
Example
Alcohol Dehydrogenase
CH3CH2OH + NAD+
CH3CHO + NADH + H+
Enz + NAD+
Enz-NAD+
Enz-NAD+
+ CH3CH2OH
Enz-NAD+ CH3CH2OH

19. PING PONG P1 P2 S1 S2 E ES1 E* E*S2 E
First product appears before second substrate binds
Serine Proteases use a Ping Pong Mechanism