Chapter 6
Mechanisms of Enzyme Action
Enzymatic Catalysis
• Activation Energy
(AE) – The energy
require to reach
transition state from
ground state.
• AE barrier must be
exceeded for rxn to
proceed.
• Lower AE barrier,
the more stable the
transition state (TS)
• The higher [TS], the
move likely the rxn
will proceed.
Enzymatic Catalysis
S  Ts  P
Transition (TS) State Intermediate
• Transition state = unstable high-energy
intermediate
• Rate of rxn depends on the frequency at which
reactants collide and form the TS
• Reactants must be in the correct orientation
and collide with sufficient energy to form TS
• Bonds are in the process of being formed and
broken in TS
• Short lived (10–14 to 10-13 secs)
Intermediates
• Intermediates are
stable.
• In rxns w/
intermediates, 2
TS’s are involved.
• The slowest step
(rate determining)
has the highest AE
barrier.
•
Formation of
intermediate is the
slowest step.
•Enzyme binding of substrates decrease activation energy
by increasing the initial ground state (brings reactants
into correct orientation, decrease entropy)
•Need to stabilize TS to lower activation energy barrier.
ES complex must not be too stable
Raising the energy of
ES will increase the
catalyzed rate
•This is accomplished
by loss of entropy due
to formation of ES and
destabilization of ES by
•strain
•distortion
•desolvation
Transition State Stabilization
• Equilibrium between ES <-> TS, enzyme drives
equilibrium towards TS
• Enzyme binds more tightly to TS than substrate
Transition
state analog
Mechanistic Strategies
Polar AA Residues in Active Sites
Common types of enzymatic
mechanisms
• Substitutions rxns
• Bond cleavage rxns
• Redox rxns
• Acid base catalysis
• Covalent catalysis
Substitution Rxns
• Nucleophillic Substitution–
O
O
R
C
R
X
C
O
X
Y
R
+ X
C
Y
Nucleophillic = e- rich
Electrophillic = e- poor
Y
• Direct Substitution
R2
R1
C
X
R3
Y
R2
R1
X C Y
R3
transition state
R2
R1
C
X
+ Y
R3
Oxidation reduction (Redox) Rxns
• Loose e- = oxidation (LEO)
• Gain e- = reduction (GER)
• Central to energy production
• If something oxidized something must be
reduced (reducing agent donates e- to
oxidizing agent)
• Oxidations = removal of hydrogen or
addition of oxygen or removal of e• In biological systems reducing agent is
usually a co-factor (NADH of NADPH)
Cleavage Rxns
• Heterolytic vs homolytic cleavage
• Carbanion formation (retains both e-)
R3-C-H  R3-C:- + H+
• Carbocation formation (lose both e-)
R3-C-H  R3-C+ + H:Hydride ion
• Free radical formation (lose single e-)
R1-O-O-R2  R1-O* + *O-R2
Acid-Base Catalysis
X
•
•
•
•
H
:B
X:
H
B
Accelerates rxn by catalytic transfer of a proton
Involves AA residues that can accept a proton
Can remove proton from –OH, -NH, -CH, or –XH
Creates a strong nucleophillic reactant (i.e. X:-)
Acid-Base Catalysis
X
H
:B
H
X:
B
carbanion intermediate
O
O
C
N
:
OH
O
H
H
:B
N
C
:
C
O
OH
HN
H
B
:B
Covalent Catalysis
•
20% of all enzymes employ covalent catalysis
A-X + B + E <-> BX + E + A
•
A group from a substrate binds covalently to
enzyme
(A-X + E <-> A + X-E)
•
The intermediate enzyme substrate complex
(A-X) then donates the group (X) to a second
substrate (B)
(B + X-E <-> B-X + E)
Covalent Catalysis
Protein Kinases
ATP + E + Protein <-> ADP + E + Protein-P
1)
A-P-P-P(ATP) + E-OH <-> A-P-P (ADP) + E-O-PO4-
2)
E-O-PO4- + Protein-OH <-> E + Protein-O- PO4-
The Serine Proteases
•
•
•
•
Trypsin, chymotrypsin, elastase, thrombin,
subtilisin, plasmin, TPA
All involve a serine in catalysis - thus the
name
Ser is part of a "catalytic triad" of Ser,
His, Asp (show over head)
Serine proteases are homologous, but
locations of the three crucial residues
differ somewhat
Substrate specificity determined by binding
pocket
Serine Proteases are structurally
Similar
Chymotrpsin
Trypsin
Elastase
Substrate binding
specificity