catalytic mechanisms. objective to understand how enzymes work at the molecular level. ultimately...
TRANSCRIPT
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