mechanisms of enzyme action. transition (ts) state intermediate transition state = unstable...
TRANSCRIPT
Mechanisms of Enzyme Action
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)
• 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 TS
• The higher [TS], the move likely the rxn will proceed.
Transition State (TS) Intermediate
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.
Catalyst stabilize the Transition State
•Enzyme binding of substrates decrease activation energy by increasing the initial ground state (brings reactants into correct orientation)
•Need to stabilize TS to lower activation energy barrier.
Common types of enzymatic mechanisms
• Substitutions rxns
• Bond cleavage rxns
• Redox rxns
Substitution Rxns
• Nucleophillic Substitution–
• Direct Substitution
C
O
XR
Y
C
O
XR
Y
C
O
YR+ X
C
R1 R2
R3 Y
CX YR1 R2
R3X
CR1 R2
X R3
+ Y
transition state
Nucleophillic = e- rich Electrophillic = e- poor
• 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:-
• Free radical formation (lose single e-)R1-O-O-R2 R1-O* + *O-R2
Cleavage Rxns
Hydride ion
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)
Polar AA Residues in Active Sites
Reactive ChargeAA Group @pH 7 Functions
Aspartate -COO- -1 Cation Binding, H+ transfer
Glutamate-COO- -1 Cation Binding, H+ transfer
Histidine Imidazole ~0 H+ transfer
Cysteine -CH2SH ~0 Binding of acyl groups
Tyrosine Phenol 0 H-Bonding to ligands
Lysine -NH3++1 Anion binding, H+
transfer
Arginine Guanidinium +1 Anion binding
Serine -CH2OH 0 Binding of acyl groups
• Accelerates rxn by catalytic transfer of a proton
• Involves AA residues that can donate or transfer protons at neutral pH (e.g. histidine)
• HB+ :B + H+
Acid-Base Catalysis
X H : B X: H B
X H : B X: H B
C
O
N
O
H H : B
C
O
OH
N
H
B
C
O
OH HN
: B
:
:
Acid-Base Catalysis
carbanion intermediate
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 KinasesATP + 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
-
Binding Modes of Enzymatic Catalysis
• Proximity Effects
• Transition State Stabilization
Proximity Effects
• In multi-substrate reactions, collecting and correct positioning of substrates important
• Increases effective concentration of substrates which favors formation of TS
• Decreases activation energy by decreasing entropy
• Increases rate of reaction > 10,000 fold• E binding to S can not be too strong or
conversion from ES to TS would require too much energy
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
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
Transition state analog
• Equilibrium between ES <-> TS, enzyme drives equilibrium towards TS
• Enzyme binds more tightly to TS than substrate
The Serine ProteasesTrypsin, 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
Substrate binding specificity