time to connect……. enzyme activity is amount of enzyme that
DESCRIPTION
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 substrateTRANSCRIPT
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
Vmax is maximum product formed at saturating substrate per minute for a given amount of enzyme,
E + S [ES] E + P
There are 2 steps in an enzyme-catalyzed reaction
1. Formation of ES2. Formation of P
Kmkcat
Km meaures the affinity of the enzyme for thesubstrate. It does not concern product formation
Kcat measures how rapidly the product is formed from ES. It does not measure binding of substrate
Time to Connect…….Kcat = k2 when ES = ET
Therefore:Vmax = kcat [E]
or
Kcat = Vmax
[E] moles Enzymemoles S per minute
= Turnover
kcat is synonymous with Turnover Number
Time to Get Efficient…….
kcat / Km =k2
k2 + k-1
k1
= k1k2
k2 + k-1
When an enzyme is maximally efficient, k2 >>k-1
= k1
At maximum efficiency, k1 controls the rate
Every collision results in ES
kcat / Km of 108 or 109 meansdiffusion control
k2
Multiple the numerator by the reciprocal of the
denominator
kcat/Km
Acetylcholine esterase
Chymotrypsin
Urease
Km(M) kcat
0.015 0.14 9.3
Catalase
0.025 1 x 104 4 x 105
0.000095 1.4 x 104 1.5 x 108
0.025 1 x 107 4 x 108
(Per second)
The rate of catalysis by Acetylcholine esterase and catalase is controlled by diffusion …
Every collision with the enzyme results in a product
Metal Ions in Catalysis- One third of all enzymes require a metal ion for catalysis
His –Zn2+
His
His
OH
CO
O
H2O
Zn 2+Polarizes H2O, making it a better
nucleophile
His –Zn2+
His
His
OH
..+ C
O
O
His –Zn2+
His
His
OH
..+ H+ + H O C
O
O
Bicarbonate
Displaces HCO3-
N N..
His 12
N
N
H
+
His 119
OO
Base
O O
O-P O
CH2
H
H
O
O
O-P-O-CH2 Base
O O
O-P
O
O
O
H
OO
Base
O O
O-P
O
O
CH2
H
H2O
O
H
H
O
O-P-O-CH2 Base
O O
O
O
H N N
POO
N
N+
..
2’,3’-cylic nucleotide
RNA Hydrolysis
O
O-P-O-CH2 Base
O O
O
O
H
N N
N
N+
O
H
HO P=O
:
+His 119
His 12
3’ end of split RNA
RNA Hydrolysis (cont.)
Insights into the Mechanism of a DigestiveEnzyme
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
Catalytic Triad of Chymotrypsin
HO CH2
C=O
N
CH..
NN
CH2
CHNC
OH
H :
CO
OCH2
CHHN
O=C:
Catalytic Site
Aspartate
Histidine
Serine
Attacking Group on Serine
O
CO
O
Asp
N NHH
His
Ser..
HO—C — CH—NH—C —CH —NH —A-A-A-A-A-A-A-A-
O R O
Hydrophobic Pocket
Chymotrypsin
O
CO
O
Asp
N NH H
His
Ser
C —CH —NH —A-A-A-A-A-A-
AA—C — CH—NH3
O R
O
Split Protein
Chymotrypsin
O
CO
O
Asp
N NH
His
Ser
C —CH —NH —A-A-A-A-A-A
O
HO
H:
Split Protein
AA—C — CH—NH3
O R
Chymotrypsin
O
CO
O
Asp
N NH
His
Ser
C —CH —NH —A-A-A-A-A-A
O
H
O
Broken Peptide Bond
AA—C — CH—NH3
O R
Chymotrypsin
ZymogensRule: Many hydrolytic enzymes are synthesized as inactive precursors. The suffix “ogen”, or the prefix pro, and prepro” designate a precursorRule: Activation requires removal of a blocking peptide by
proteolytic cleavageTrypsin
COOH
S - S S - S
-chymotrypsin (active)
Chymotrypsin
COOH
S - S S - S
-chymotrypsin (active)
COOH
S - S S - S
Chymotrypsinogen (inactive)
More than One Substrate…..
E + S1 + S2
RandomE + S1 then S2
E + S2 then S1E-S1-S2 E + P1 + P2
Example Glucose + ATP
E + Glucose E-GlucoseE-Glucose-ATPor
E + ATP E-ATP
+ ATP
+ Glucose
Hexokinase
More than One Substrate…..
E + S1 + S2ORDERED
E + S1 E-S1 E-S1-S2E + S2 No Reaction
Example Alcohol Dehydrogenase
CH3CH2OH + NAD+ CH3CHO + NADH + H+
Enz + NAD+ Enz-NAD+
Enz-NAD+ + CH3CH2OH Enz-NAD+ CH3CH2OH
PING PONG
First product appears before second substrate binds
S1
E ES1
P1
E*
S2
E*S2
P2
E
Serine Proteases use a Ping Pong Mechanism