Biochemistry
Lecture 8
Why Enzymes?
•
•
•
•
Higher reaction rates
Greater reaction specificity
Milder reaction conditions
Capacity for regulation
COO
-
COO
NH2
O
OH
COO
OH
COO
Chorismate
mutase
COO
OOC
O
NH2
-
-
O
COO
COO
OH
-
• Metabolites have
many potential
pathways of
decomposition
• Enzymes make the
desired one most
favorable
Enzymatic Substrate Selectivity
OH
H
-
H
+
NH3
OOC
-
H
-
+
NH3
No binding
+
NH3
OOC
OOC
OH
HO
OH
H
H
H
NH
CH3
Example: Phenylalanine hydroxylase
Binding but no reaction
How to Lower
Enzymes organizes
reactive groups into
proximity

G ?
How to Lower

G ?
Enzymes bind transition states best
How is TS Stabilization
Achieved?
– acid-base catalysis: give and take protons
– covalent catalysis: change reaction paths
– metal ion catalysis: use redox cofactors, pKa shifters
– electrostatic catalysis: preferential interactions with TS
End result? Rate enhancements of 105 to 1017!
How is TS Stabilization
Achieved?
– covalent catalysis: change reaction paths
O
O
H3C
H3C
H2O
O
CH3
O
O
O
O
H3C
slow
O
+
O
+
O + 2H
H3C
O
CH3
fast
O
+
N
CH3
..
N
..
H
O
O
-
H
+
H3C
O
-
Enzyme Kinetics
• Kinetics is the study of the rate at which
compounds react
• Rate of enzymatic reaction is affected by
– Enzyme
– Substrate
– Effectors
– Temperature
How to Do Kinetic
Measurements
What equation models this
behavior?
Michaelis-Menten Equation
Meaning of Vmax and Km
Simple Enzyme Kinetics
• The final form in case of a single substrate is
kcat [ Etot ][S ]
v
K m  [S ]
• kcat (turnover number): how many substrate
molecules can one enzyme molecule convert per
second
• Km (Michaelis constant): an approximate measure of
substrate’s affinity for enzyme
• Microscopic meaning of Km and kcat depends on the
details of the mechanism
Two-substrate Reactions
• Kinetic mechanism: the order of binding of
substrates and release of products
• When two or more reactants are involved, enzyme
kinetics allows to distinguish between different
kinetic mechanisms
–
–
Sequential mechanism
Ping-Pong (Double Displacement) mechanism
Distinguishing Mechanism
Ternary Complex
Ping-Pong
Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity
• Irreversible inhibitors (inactivators) react with the enzyme
- one inhibitor molecule can permanently shut off one enzyme molecule
- they are often powerful toxins but also may be used as drugs
• Reversible inhibitors bind to, and can dissociate from the enzyme
- they are often structural analogs of substrates or products
- they are often used as drugs to slow down a specific enzyme
•
Reversible inhibitor can bind:
– To the free enzyme and prevent the binding of the substrate
– To the enzyme-substrate complex and prevent the reaction