Transcript Lecture_5a_ Catalysis . ppt - University of Massachusetts

Enzyme Catalysis
Bill Royer
Office: LRB 921
Phone: x6-6912
Enzymes have spectacular abilities to accelerate chemical reactions –
often by factors of 106-1014 over non-catalyzed reactions. In this
lecture, we will briefly discuss some of the strategies used by enzymes
to achieve such remarkable rate increases.
I. Transition state theory
II. Mechanisms of catalysis
Acid-base catalysis - Ribonuclease A
Metal ion catalysis - Hammerhead Catalytic RNA
Covalent catalysis - Chymotrypsin
I. Transition state theory
Consider the reaction A + B  P + Q
A+B
K‡
‡
k'

P+ Q
where A + B react through transition state, X‡, to form products P + Q. K‡ is the
equilibrium constant between A + B and X‡ and k' is the rate constant for
conversion of X‡ to P + Q.
‡
G ‡
G
A+B
G react ion
P+ Q
Reaction coordinate
T he minimum energy pat hway of t he react ion
is shown in t he react ion coordinate, or
t ransition st at e diagram, at left. Chemical
conversion ofA + B t o P + Q proceeds
t hrough a t ransition st ate
‡ which is the
least stable (least probable, highest free
energy) species along the pathway.
Molecules that achieve the activation energy,
G‡ , can go on t o react while molecules that
fail t o achieve t he transit ion stat e fall back t o
t he ground state.
The transition state, X‡, is metastable. (Unlike a reaction intermediate, the transition
state has only a transient existence, like a pebble balanced on a pin. By definition, a
transition state cannot be isolated.) The transition state can be thought of as sharing
some features of the reactants and some features of the products. That is, some
bonds in the substrate are on their way to being broken and some bonds in the
product are partially formed.
The transition state, X‡, is in rapid equilibrium with reactants
with equilibrium constant K‡.
‡

K‡ 
[A] [ B]
G‡, the activation energy, is the difference in Gibbs free energy between the transition
state, X‡, and the reactants. Since K‡ is an equilibrium constant, the now familiar
equation applies:
‡
‡
-RT lnK = G
where T is the absolute temperature in degrees Kelvin (°C + 273) and R is the gas constant
(1.98 cal / mol / degree). In other words, the frequency with which reactants achieve the
transition state is inversely proportional to the activation energy barrier between the two.
The observed rate of the reaction, kobs, will be a
function of the concentration of the reactants, the rate
of conversion of X‡ to P + Q, k', and will decrease
exponentially with an increase in G‡.
‡
k obs = k' e
-G / RT
[A][B]
Thus, the smaller the difference in free energy of the reactants and the transition state, the faster
the reaction proceeds. Enzymatic rate accelerations are achieved by lowering the activation
barrier between reactants and the transition state, thereby increasing the fraction of
reactants able to achieve the transition state. Enzymes reduce the activation barrier by
destabilizing the ground state of enzyme-bound substrates and products, by stabilizing the
transition state, and/or by introducing a new reaction pathway with a different transition state that
has a lower free energy.
‡
Uncataly zed
Enzymes accelerate react ions by lowering t he
energy barrier bet ween reactants and product s.
‡
Gcat
G‡ = G‡uncat al yzed -G‡catalyzed
G
A+B
Cataly zed
A+B
P+Q
P+ Q
Reaction coordinate
Alt hough less energy is required to form t he
t ransit ion stat e in t he cat alyzed react ion, the
ground st at es of t he free substrates and product s
remain the same. T he kinet ic barrier is lowered
by the sam e ext ent for t he forward and reverse
reactions. Consequent ly,
a catal ys t acce le rate s
the re acti on with ou t affe cti n g i ts e qu il ibri
.
If a catalyst lowers the activation barrier by G‡, the rate of the reaction is enhanced
by the factor e G‡/RT. Consequently, a ten-fold rate enhancement requires that G‡ =
1.36 kcal/mole, less than the energy of a single hydrogen bond.
(G‡ = RTln10 = 1.98 x 10-3 kcal/mol*K x 298K*ln(10) = 1.36 kcal/mol)
Imaginary enzyme ("stickase") designed to catalyze "cleavage" (breaking) of a metal stick
(Nelson & Cox, Lehninger Principles of
Biochemistry, 3rd ed., 2000)
For a reaction that involves several steps, each step will have a corresponding
transition state.
k1
AI
A‡
G
I‡
kk11<> kk22
I
k1
I
k2
Reaction coordinate

P
kk11 >< kk22 If t he formation of I, an intermediat e, from A
A
A
k2
P
P
slower t han the formation of P from 1I (k 2 < k
t he activat ion barrier for the first step must b
higher t han the act ivation barrier for t he seco
step (t hick line). If1k is much slower t han
2 k
conversion of A t o I is the rat e-det ermining s
for t he react ion. T hat is, t he overall reaction
proceeds at a rate t hat can be no fast er t han
1 k
Conversely, if formation of P from I is much
slower t han form at ion of I from A2 (k 1< k ),
act ivation barrier for t he second st ep is highe
(thin line) and format ion of P from I is
rat e-det ermining.
II. Mechanisms of catalysis
A. Acid-base catalysis
Specific acid or base catalysis - Reaction rate is directly proportional to [H+] or [OH-].
Example: Alkaline hydrolysis of RNA
General acid or base catalysis - Reaction rate is proportional to [Bronsted acid] or
[Bronsted base]
Bronsted acid - species that can donate protons
Bronsted base - species that can combine with a proton
Specific Base Catalysis
General Base Catalysis
Rate
Rate
pH 7.3
pH 7.0
pH 7.3
pH 7.0
[I midazole buf f er]
[I midazole buf f er]
Amino acids side chains with pKa's in the neutral pH range can
function as Bronsted acids/bases
Amino Acid
Aspart ic acid
pK a
3.90
-COOH
COO-
O
H C CH
NH3+
2
C
O-COOH
COOGlutamic acid
4.07
H C CH2
NH3+
O
CH2
C
O-
COOHistidine
6.04
H C CH2
NH3+
imidazole
N
N
COOCy st eine
8.33
H C CH2
sulf hy dryl
SH
NH3+
phenol
COOTyrosine
10. 13
H C CH2
OH
NH3+
-amino
COOLysine
10. 79
H C CH2
NH3+
CH2
CH2
NH +
3
Biologically important
nucleophilic groups:
Nucleophilic
form
Hydroxyl group
R-OH
R-O:
Sulfhydryl group
R-SH
R-S: -
+ H+
Amino group
R-NH3+
R-NH2
+ H+
R
Imidazole group
HN + NH
+ H+
R
HN
N:
+ H+
Biologically important
electrophiles:
H+
Protons
Mn+
Metal Ions
C=O
Carbonyl carbon
R-NH2
+ C=O
Adapted from
Voet & Voet,
Biochemistry
Ribonuclease A
An example of concerted acid-base catalysis - reaction subject to both
general acid and general base catalysis
5'... O
5'... O
O Pyri midi ne
5'... O
O
O Pyri midi ne
Pyri midi ne
H2 O
O OH
O P O
O
O
O O
P
O O-
2',3'-Cyclic phosphat e
Base
H
O OH
O P O
O
3' phosphate
HO
O
Base
OHOH
3'...O OH
RNase A (124 residues, mw 13.7 kd) is a digestive enzyme secreted by the pancreas
that catalyzes hydrolysis of phosphodiester backbone of RNA. In first step of the
reaction, cleavage of the bond between phosphorous and the 5' oxygen generates one
2',3'-cyclic phosphate terminus and one 5'-OH. In the second step, water reacts with
the cyclic phosphate to yield a 3' phosphate. The 2',3' cyclic phosphate can be
isolated because it forms more rapidly than it hydrolyzes.
First Step: 2’3’ cyclic nucleotide produced. His 12 is general base, His 119 is general acid
Transesterif ication
5'... O
5'... O
Charge
A stabilizat ion
Lys 41
O
O Pyri midi ne
His 12
Nucleophilic att ack
of 2' O on phosphate O O
His 119
HN
NH+
O P O
O
NH+
:N
H
O
O
O
O
Acid protonates
5' leav ing group 3'... O OH
Pyri midi ne
O O
P
O OHO
P
Base
O
+H 3 N
O
Base abst ract s
proton f rom 2' OH
5'... O
O
O
O
Base
3'... O OH
Trigonal bipy ramidal transition state
Base
3'... O OH
Intermediate
Second Step:
Hydrolysis of 2',3' cyclic phosphate intermediate
5'... O
Acid protonates
2' OH leav ing group
O
NH+
Pyri midi ne
5'... O
O Pyri midi ne
His 119
His 12
HN
Base abst ract s
proton f rom H
2 O
O O
P
-O O
H
O
His 12 is general acid, His 119 is general base
:N
H
NH+
Nucleophilic att ack of
H 2 O on phosphate
O OH
O P O
O
3' phosphate
Proposed mechanism of RNase A catalysis. The unionized form of His 12 accepts a
proton from the 2' OH which enhances its nucleophilicity. The protonated form of His 119
begins to donate its proton to the 5' O, and the 2'O begins to form a bond with P to form a
pentacoordinate transition state. The negative charge that develops is stabilized
electrostatically by the nearby positively charged side chain of lysine 41. The bond between
P and the 5'-O breaks when the proton from histidine 119 is completely transferred. At the
same time, a bond between P and the 2'-O becomes fully formed, producing the 2',3'-cyclic
intermediate. Hydrolysis of the cyclic intermediate is a reversal of the first stage with H2O
replacing the 5'-O component that was removed. Histidine 12 is now the proton donor and
histidine 119 is the proton acceptor.
Geometry of the pentacovalent transition state. The central
phosphorus atom is transiently bonded to 5 oxygen atoms. Three
oxygens are coplanar with the phosphorus. The oxygen atoms of the
leaving group is at one apex, and the oxygen atom of the attacking group
is at the other apex of the trigonal bipyramid (in-line attack).
Evidence for RNase A mechanism
pH dependence of Vmax/KM for RNase A catalyzed
hydrolysis of cytidine-2',3'-cyclic phosphate. Bell shaped
curve suggests a catalytic role for functional groups with
pK's of 5.4 and 6.4, consistent with histidines.
Crystal structure of RNase A complex with cytidine 2'3'cyclic phosphate intermediate. Shows histidines and lysine
appropriately positioned in the active site. Note hydrogen
bonding interactions between cytosine and threonine 45
that confer substrate specificity.
Chemical modification. Iodoacetate alkylates histidine 119 or histidine 12 but not both in the
same molecule. Alkylation of either histidine eliminates catalysis. Complex formation with
substrate or competitive inhibitors protects histidines from modification.
B. Metal ion catalysis
1. Water ionization. A metal ion's charge makes its bound water molecules more acidic than
free H2O and therefore a source of OH- ions even below neutral pH (Metal ions have been
called "Super acids").
Mg 2+ H2O
Mg 2+ OH - + H +
pKa = 11
2. Charge shielding - metal ions can have charge > +1.
3. Oxidation-Reduction
The Hammerhead Catalytic RNA
The hammerhead ribozyme, like RNase A,
catalyzes a transesterification reaction to cleave
the phosphodiester backbone of substrate
RNAs yielding products with 5' hydroxyl and
2'3'cyclic phosphate termini. Unlike the RNase
A-catalyzed reaction, the hammerhead reaction
does not proceed through hydrolysis of the 2',3'
cyclic phosphate.
G C 5'
Substrat e
GC
Ribozy me
A U
CG
cleav age site
A U
A
C
A
A
AGGAU
U GGCC G
U GCCGG
UCCUGGG5'
C
A
G AGU
U
Hammerhead Catalytic RNA
The hammerhead ribozyme obviously has no amino acid side chains to carry out proton
transfer and charge-shielding functions. RNAs are, however, capable of binding metal ions
with high specificity and affinity and the hammerhead ribozyme appears to make use of
metal ions to carry out both charge shielding and proton transfer functions.
C. Covalent catalysis - Transient formation of a catalyst-substrate covalent bond
-Provides an alternative reaction pathway, with two lower energy transition states
1. A nucleophile (electron-rich group with a strong tendency to donate electrons to an
electron-deficient nucleus) on the enzyme displaces a leaving group on the substrate,
forming a covalent bond.
2. The enzyme substrate bond decomposes to form product and free enzyme.
-Covalent catalyst must be a good nucleophile and a good leaving group - highly mobile
electrons (imidazole of His, thiol of Cys, carboxyl of Asp, hydroxyl of Ser).
Chymotrypsin, 25 kd serine protease, catalyzes hydrolysis of proteins in the small
intestine. Chymotrypsin catalyzes hydrolysis of esters as well as peptide bonds which has
been useful for analysis of the catalytic mechanism, although not physiologically relevant.
Model reaction in w hich hydrolysis of acyl-enzyme intermediate is slow
O
O
CH 3 C o
NO2 + Chymotrypsin
f ast
p-Nitrophenylacetate
-O
NO2
p-Nitrophenylate
Formation of the acyl-enzyme intermediate
occurs during the initial rapid phase and
slower hydrolysis (deacylation) of the acylenzyme intermediate occurs during the
second, slower phase.
chymotrypsin
+
CH3 C
chymotrypsin
Acyl-enzyme intermediate
H2 O
slow
H+
O
CH3 C
O-
+
Acetate
[p-Nitrophenylate], M
32M
30
24M
20
16M
10
8M
2
4
6
8
Time (min)
10
12
T he plot at left shows t he concentration of
p-nitrophenol produced as a funct ion of t ime in
reactions cont aining different concent rations
of chymot rypsin and a large excess of
p-nirophenylacet at e. An initial rapid phase
("burst") is followed by a slower phase. T he
size of t he init ial burst is proportional to t he
enzyme concentrat ion. "Burst " kinet ics
provide evidence for a stable, enzym e-linked
int ermediate.
chymotrypsin
First stage in peptide bond hydrolysis: acylation. Hydrolysis of the peptide bond starts with an
attack by the oxygen atom of the Ser195 hydroxyl group on the carbonyl carbon atom of the
susceptible bond. The carbon-oxygen bond of this carbonyl group becomes a single bond, and
the oxygen atom acquires a net negative charge. The four atoms now bonded to the carbonyl
carbon are arranged as a tetrahedron. Transfer of a proton from Ser195 to His57 is facilitated
by Asp102 which (i) precisely orients the imidazole ring of His57 and (ii) partly neutralizes the
positive charge that develops on His57 during the transition state. The proton held by the
protonated form of His57 is then donated to the nitrogen atom of the peptide bond that is
cleaved. At this stage, the amine component is hydrogen bonded to His57, and the acid
component of the substrate is esterified to Ser195. The amine component diffuses away.
Oxyanion
hole
Second stage in peptide hydrolysis: deacylation. The acyl-enzyme intermediate
is hydrolyzed by water. Deacylation is essentially the reverse of acylation with
water playing the role as the attacking nucleophile, similar to Ser195 in the first
step. First, a proton is drawn away from water. The resulting OH- attacks the
carbonyl carbon of the acyl group that is attached to Ser195. As in acylation, a
transient tetrahedral intermediate is formed. His57 then donates a proton to the
oxygen atom of Ser195, which then releases the acid component of the substrate,
completing the reaction.
Oxyanion
hole
Chymotrypsin catalytic triad – Ser195/His57/Asp102 located at the active site
by x-ray crystallography.
An important stabilizing feature of the interaction between enzymes and their
substrates, is transition state binding. In fact, most enzyme active sites are
organized such that binding to the transition state is preferred over binding to
either substrates or products. The active site of chymotrypsin is arranged to
stably interact with the negatively charged carbonyl oxygen of the tetrahedral
intermediate – this part of the active site is referred to as the “oxyanion hole”.
Mechanism of Protein Splicing:
The protein splicing pathway
consists of four nucleophilic
displacements. X represents the S or
O atom of the Cys/Ser/Thr
sidechains.
From: Perler, FB (1998) Cell 92, 1-4