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Objective
To understand how enzymes work at the molecular
level.
Ultimately requires total structure determination, but can learn much through biochemical
analysis.
To Be Explained
• Specificity– For specific substrates– Amino acids residues involved
• Catalysis– Mechanisms– Amino acids involved/Specific role(s)
Enzyme Binding Sites
• Active Site:– Substrate Binding Site + Catalytic Site
• Regulatory Site: – a second binding site, – Binding by regulatory molecule affects the active site
• alter the efficiency of catalysis • improve or inhibit
General Characteristics
• Three dimensional space• Occupies small part of enzyme volume• Clefts or crevices
• Ligands (substrate or effector) bound by multiple weak interactions
• Specificity depends on precise arrangement of atoms in active site
Identification and Characterization of Active
Site
• Structure: size, shape, charges, etc.
• Composition: identify amino acids involved in binding and catalysis.
Binding or Positioning Site(Trypsin)
NH CH C NH
O
N C
complementary binding or posit ioning site
"SPECI FI CI TY"_
+
arginine or lysine
"long + side chain"
H2O
Binding or Positioning Site(Chymotrypsin)
NH CH C NH
O
N C
"aromatic side chain"
"SPECI FI CI TY"
complementary binding or posit ioning site
phenylalaninetyrosinetryptophan
Hydrophobic Pocket
H2O
O
Model Substrates(Chymotrypsin)
H2O(ROH)
NH CH CN
R
NH
O
C
acyl transfer to H2Oaromaticside chain
peptide bond
Peptide Chain?
All Good Substrates!
H3N CH C NH
O
C
R
NH CH CH3N
R
NH2
O
(or -OCH3)
or
H3N CH C
R
NH2
O
(or -OCH3)
or
ConclusionBulky Hydrophobic Binding Site
CH C X
O
Y
"Hydrophobic Acyl Group Transferase"
= hydrophobic posit ioning group
X,Y = various
Arginase
H2N
C
NH
(CH2)3
CH COOH3N
NH2H2O
NH3
(CH2)3
CH COOH3N
H2N
C
O
NH2
+
+
-+
ureaornithinearginine
+ -
+
Good Competitive Inhibitors
NH3
NH
NH3
(CH2)3
CH COOH3N
NH3
(CH2)4
CH COOH3N
O
(CH2)2
CH COOH3N
CH
NH2
-+
(
ornithine
(+
-
++
+
-
(
canavaninelysine
+
Poor Competitive Inhibitors
All Three Charged Groups are Important
NH3
(CH2)3
CH2H3N
NH3
(CH2)3
H2C COO
CH3
(CH2)3
CH COOH3N+ -
++
+ -
a-aminovaleric acid putrescine
(l,4-diaminobutane)4-aminovaler ic acid
Identifying Active Site Amino Acid Residues
• Covalent modification of residues– Inactivation of enzyme
• Site directed mutagenesis– Inactivation of enzyme
Mechanisms of Catalysis
• Acid-base catalysis
• Covalent catalysis
• Metal ion catalysis
• Proximity and orientation effects
• Preferential binding (stabilization) of the transition state
Figure 11-10
Ribonuclease A
N
N
O
O
OH
O
O
CH2
O
PO O
O
N
N
N
N
O
NH2
OH
CH2OP
O
O
O
PO O
O
Adenosine
UridineRibonuclease A
Covalent Catalysis(Nucleophilic catalysis)
(Principle)
Involves a transient covalent bond between the enzyme and the substrate
Usually by the nucleophilic attack of the substrate by the enzyme
Covalent Catalysis(Principle)
SlowH2O + A–B ——> AOH + BH
A-B + E-H ——> E-A + BHE-A + H2O ——> A-OH + E-H
Fast
NOTE: New Reaction Pathway
Covelent Catalysis
The Schiff Base
• Metalloenzymes: tightly bound metal ions– Catalytically essential– Fe2+, Fe3+, Cu2+, Mn2+, and Co2+
• Metal-activated enzymes: loosely bound metal ions (from solution or with substrate)– Structural metal ions: – Na+, K+, and Ca2+
• Both: Mg2+ and Zn2+
Metal Ion Catalysis
Carbonic Anhydrase
Carbonic Anhydrase
Proximity and Orientation Effects
Rate of a reaction depends
on:
• Number of collisions• Energy of molecules• Orientation of molecules• Reaction pathway (transition state)
Page 336
Intramolecular Reaction of Imidazole with p-Nitrophenylacetate
(Intramolecular)
Intramolecular Rate = 24x Intermolecular Rate
Preferrential Binding of Reaction Intermediate
• Stabilize Transition State– Electrostatic stabilization of developing charge
– Relief of induced bond angle strain
– Enhancement of weak interactions between enzyme and intermediate.
Convergent Evolution
Substrate Specificity
Mechanism of Chymotrypsin
CT CH2 OH O2N O C CH3
O
CT CH2 O C CH3
O
CT CH2 OH
OO2N
CT CH2 O C CH3
O
HO C CH3
O
"rate limiting"
++ H2O
+fast
+
fast
p-nitrophenolate