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What is an enzyme?
Enzyme is protein or nucleic acid.
Enzyme Catalysis
•
Chemical reactions
– breaking, forming and rearranging bonds.
thermodynamic aspects
•
Specificity
– Dictated by the enzyme active site.
– Some active sites allow for multiple substrates.
•
Cofactors
– Amino acid side chains have a limited chemical repertoire.
– Vitamin derivatives, metals (minerals) can bind as co-substrates
or remain attached through multiple catalytic cycles
Active Site
•
Small relative to the total volume
of the enzyme.
•
Usually occur in clefts and
crevices in the protein. Excluding
solvents which would otherwise
reduce the catalytic activity of the
enzyme.
•
Amino acids and cofactors are
held in precise arrangement with
respect to structure of the
substrate.
•
The specificity of substrate
utilization depends on the
defined arrangement of the
atoms in the enzyme active site
(complements the structure of
the substrate molecule).
Crystal Structure of DNA Ligase
1
2
k2  k1  kcat  k2
kcat  E0   Vmax
v
 E0  S  kcat
KM  S 
3
Relation to thermodynamics
H  U pot  pV
G  H  T S
G  H  TS
G 0   RT ln K eq
Boltzmann rules!
0
PB
 K eq  eG / RT
PA
k 
k BT G‡ / RT
e
h
k = rate constant
Keq = equilibrium constant
G ‡
PA
Thermodynamic cycles and free energies
k2  k1 

E + S‡
(K‡bind)
 ES 
1/KM
KM 
‡
k1
k1
water
G

K=1
E+S
Free energy diagrams - comparison to uncatalyzed reaction
in water is essential
kcat  k2

wat
knon
E+S
PB
ES‡

E+S
E + S‡
E+S
kcat
 ES 
Gbind

Gcat
ES‡
G (kcal/mol)

Genz
enzyme

Genz
kcat / K M


kcat / K M  k non K bind
 K bind

kcat / K M
k non



Gbind
 Gbind  Gcat
 Gwat

Gwat
‡

( Gbind
)
0

Gcat
E+S
EP
ES
E+P
reaction coord.
4
How do enzymes work?
Catalytic strategies - chemical ”tricks” in catalysis:
•
Oversimplified picture of enzyme catalysis
Catalytic strategies - chemical ”tricks” in catalysis:
Covalent catalysis
We will not be concerned with ”chemical” effects
(they are more or less trivial !)
– The active site contains a reactive group, usually a powerful
nucleophile that becomes temporarily covalently modified in the
course of catalysis.
•
General acid-base catalysis
O
HO
– A molecule other than water plays the role of a proton donor or
acceptor.
•
Metal ion catalysis
2-
O P O3
H
H
O
-
O
G lu 1 6 5
O
HO
H
O
-
2-
OPO3
H
O
(aq)
– Metal ions can serve as electrophilic catalysts, stabilizing negative
charge on a reaction intermediate.
5
Origin of the catalytic effect in enzymes
Classic transition state stabilization – many examples
serine proteases
Hypotheses:
kcat / knon  109
 electrostatic stabilization
His
Asp
O
 strain and distorsion
O
-
H N
+
no triad
kcat
/ knon  103
N
Ser
H
 entropy: proximity and alignment
O
(Carter & Wells, 1988)
-
R
 desolvation
O
H N
oxyanion hole still there
R'
 steric effect is small
 quantum effects: tunnelling
Reaction Free Energy and Reorganization Free Energy
Reduction: two different ways of stabilizing the TS
Reorganization energy and the preorganization effect
uncatalyzed
reaction step
Marcus formula:
G  
G*
reduction of G0

( G 0   ) 2
4
G*
reduction of 
-
-
preorganization
=
reduction of 
-
something to think about:
is this why enzymes are big?
6
Enzyme catalyzed proton abstraction from
carbon acids (keto-enol isomerization)
What is required for catalysis of this type of reaction?
G
(kcal/mol)
+30
+26
O
O
C
C
C
C
Lys
N
H
O
H
C
C
H
CH3
H
C
CH3
H
O
HO
O
B
BH
H
KSI stabilizes the ionic intermediate by polar interactions
GlxI
C
TIM
B
KSI
citrate synthase
O
H
H
Me2+
O
His
OH
H
H
NH
OH
O
H
O
HO
B
H
C
GS
O
O
N
Tyr
Asp
C
C
H
C
SCoA
H
B
H
B
These enzymes work by electrostatic TS stabilization
water
Y57
Y16
C
H
H
enzyme examples: triosephosphate isomerase (TIM)
rubisco
citrate synthase
glyoxalase I (GlxI)
mandelate racemase
enolase
glucarate dehydratase
ketosteroid isomerase (KSI)
•
•
•
Y32
D103
HO
O
HO
H
OH
O
C
NH3+
NH
Usually very high pKa
O
H
His
acetone (uncatalyzed)
expt
O
wat
H
enzyme
HO
H
D40
O
H
N
W120
ketosteroid isomerase (KSI)
7
Origin of the catalytic effect: hypotheses
Origin of the catalytic effect: hypotheses
 Desolvation:
• When substrate binds to the enzyme surrounding water in
solution is replaced by the enzyme. This makes the substrate
more reactive by destablizing the charge on the substrate.
•
 Strain and Distortion:
 When a substrate bind to the enzyme, it may induce a
conformational change in the active site to fit to a transition
state.
Expose a water charged group on the substrate for interaction
with the enzyme.
 Frequently, in the transition state, the substrate and the
enzyme have slightly different structure (strain or distortion)
and increase the reactivity of the substrate.
• Also lowers the entropy of the substrate (more ordered).
cyclic phosphate ester
Acylic phospodiester
108
Rate:
1
But this example refers to uncatalyzed reaction rates of different substrates!
But ionized groups become neutral in a hydrophobic environment!
Jencks entropy hypothesis: E ‘freezes’ S upon binding
The strain hypothesis: ground state destabilization
‡


Gcat
 H cat
 H w
enz


Gcat
 Gw  Gcat
 T S   T Sbind


Genz
 Gw  Genz
 H bind
transition state
Energy
wat
strain
assumption:
T S w
Gw
reactants
Generally difficult since enzymes are ”soft” and strain energy will dissipate:
H w

Gcat
Gbind
H bind
enzyme
substrate
T Sbind
 v / v0 
S   k ln  enz enz
0 
 vw / vw 

Genz
wat

T S enz
H

H cat
 H w

enz
enz
ES
E+S
TS
reactants
enzyme
E+S
ES
ES‡
8
Wolfenden statistics:
9