Lecture 5a Catalysis

8/2/2019 Lecture 5a Catalysis

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Enzyme CatalysisBill Royer

Office: LRB 921Phone: x6-6912

I. Transition state theory

II. Mechanisms of catalysis

Acid-base catalysis - Ribonuclease A

Metal ion catalysis - Hammerhead Catalytic RNA

Covalent catalysis - Chymotrypsin

Enzymes have spectacular abilities to accelerate chemical reactions often by factors of 10 6-10 14 over non-catalyzed reactions. In thislecture, we will briefly discuss some of the strategies used by enzymesto achieve such remarkable rate increases.


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I. Transition state theory

Consider the reaction A + B P + Q

where A + B react through transition state, X , to form products P + Q. K is theequilibrium constant between A + B and X and k' is the rate constant forconversion of X to P + Q.

A + B P + QK k'

A + B

P + Q

G reaction

G

G

Reaction coordinate

The minimum energy pathway of the reactiois shown in the reac tion coordinate, o r

trans ition state diagram, at left. Chemicalconversion of A + B to P + Q proceedsthrough a transition state which is theleast stable (least probable, highest freeenergy) species along the pathway. Molecules that ac hieve the ac tivation energy,

G , c an go on to react while molecules thatfail to achieve the transition state f all back tothe ground state.

The transition state, X , is metastable. (Unlike a reaction intermediate, the transitionstate has only a transient existence, like a pebble balanced on a pin. By definition, atransition state cannot be isolated.) The transition state can be thought of as sharingsome 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 theproduct are partially formed.


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The transition state, X, is in rapid equilibrium with reactants

with equilibrium constant K. K

[A] [ B]

-RT lnK = G

G, the activation energy, is the difference in Gibbs free energy between the transitionstate, X, and the reactants. Since K is an equilibrium constant, the now familiar equation applies :

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 thetransition state is inversely proportional to the activation energy barrier between the two.

The observed rate of the reaction, k obs , will be afunction of the concentration of the reactants, the rateof conversion of X to P + Q, k', and will decreaseexponentially with an increase in G .

k = k' eobs- G / RT [A][B]


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Thus, the smaller the difference in free energy of the reactants and the transition state, the fasterthe reaction proceeds. Enzymatic rate accelerations are achieved by lowering the activationbarrier between reactants and the transition state, thereby increasing the fraction of reactants able to achieve the transition state. Enzymes reduce the activation barrier bydestabilizing the ground state of enzyme-bound substrates and products, by stabilizing thetransition state, and/or by introducing a new reaction pathway with a different transition state thathas a lower free energy .

A + B

P + Q

G

Reaction coordinate

Gcat

A+B P+Q

Uncatalyzed

Catalyzed

Enzymes accelerate reactions by lowering theenergy barr ier between reactants and products.

G = G - G

Although less energy is required to form thetransition state in the catalyzed reaction, theground states of the free substrates and productsremain the same. The kinetic barrier is lowered

by the same extent for the for ward and reversereactions. Consequently, a catalyst acceleratesthe reaction without affecting its equilibrium .

uncatalyzed catalyzed

If a catalyst lowers the activation barrier by G, the rate of the reaction is enhancedby 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 = RT ln10 = 1.98 x 10 -3 kcal/mol*K x 298K* ln(10) = 1.36 kcal/mol)


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(Nelson & Cox, Lehninger Principles of Biochemistry , 3rd ed., 2000)

Imaginary enzyme (" stickase ") designed to catalyze "cleavage" (breaking) of a metal stick


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A

P

G

Reaction coordinate

A I

I

k1A Pk2I

k1 k2

If the formation of I, an intermediate, from A islower than the formation of P from I (k < k the activation barrier for the first step must behigher than the act ivation barr ier for the seconstep (thick line). If k is much slower than k conversion of A to I is the rate-determining stefor the react ion. That is, the overall react ion

proceeds at a rate that can be no faster than k Conversely, if formation of P from I is muchslower than formation of I from A (k < k ),ac tivation barrier for the second step is higher (thin line) and formation of P from I israte-determining.

1 2

2 1

1 2

1

A k1 k2

I P

For a reaction that involves several steps, each step will have a correspondingtransition state.

k 1 < k 2k 1

> k 2


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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

[Imidazole buffer]

R a

t e

[Imidazole buf fer]

R a

t e

Specific Base Cata lysis Genera l Base Catalysis

pH 7.3

pH 7.0

pH 7.3

pH 7.0


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H C

NH

COO

CH CO

O-2

-

+3

Aspartic acid

Amino Acid pK a

3.90

-COOH

H C

NH

COO

CH CO

O-2

-

+3

Glutamic acid 4.07

-COOH

CH 2

H C

NH

COO

CH 2

-

+3

Histidine 6.04

imidazole

N

N

H C

NH

COO

CH SH2

-

+3

Cysteine 8.33

sulfhydryl

H C

NH

COO

CH 2

-

+3

Tyrosine 10.13

phenol

OH

H C

NH

COO

CH CH2

-

+3

Lysine 10.79

-amino

CH 2 2 NH 3+

Amino acids side chains with pKa's in the neutral pH range canfunction as Bronsted acids/bases


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Biologically importantnucleophilic groups:

Hydroxyl group R-OH R-O : + H+

-

Sulfhydryl group R-SH

Adapted from

Voet & Voet, Biochemistry

R-S : - + H +

R-NH 3+ R-NH 2 + H +Amino group

HN NH+

R

HN N:

R

+ H +Imidazole group

Nucleophilicform

R-NH 2 + C =O

Biologically importantelectrophiles:

C=OH+ Mn+

Protons Metal IonsCarbonyl carbon


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O

OHOH

Base

O

O O

O5'...

P

Pyrimidine

OHO

Base

3'...

O O -

2',3'-Cyclic phosphate

O

O OHP OO

O

PyrimidineO5'...

HH O2

O

O OHP OO

O

PyrimidinO5'...

3' phosphate

O OH

Ribonuclease AAn example of concerted acid-base catalysis - reaction subject to bothgeneral acid and general base catalysis

RNase A (124 residues, mw 13.7 kd) is a digestive enzyme secreted by the pancreasthat catalyzes hydrolysis of phosphodiester backbone of RNA. In first step of thereaction, cleavage of the bond between phosphorous and the 5' oxygen generates one2',3'-cyclic phosphate terminus and one 5'-OH. In the second step, water reacts withthe cyclic phosphate to yield a 3' phosphate. The 2',3' cyclic phosphate can beisolated because it forms more rapidly than it hydrolyzes.


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O

OO

O5'...A

P

O

O

O

O OH

Ba se

3'...

O

O

O O

O5'...

P OOO

Pyrimidine

O

O OH

Ba se

3'...

H Base abstractsproton f rom 2' OH

NHHN +

His 119

Acid protonates5' leaving group

NH:N +

His 12

Chargestabilization

Trigonal bipyramidal transition s tate

O

O O

O5'...

P

Pyrimidine

O

O OH

HO

Ba se

3'...

O O -

Intermediate

H N3

Lys 41

+

Transesterification

Nucleophilic attackof 2' O on phosphate

First Step: 23 cyclic nucleotide produced. His 12 is general base , His 119 is general acid

Hydrolysis of 2 ',3' cyclic phosphate intermediate

O

O O

O5'...

P

Pyrimidine

O O- HO

HNH:N +

His 119

Base abstractsproton f rom H O2

Nucleophilic attack ofH O on phosphate2

NHHN +

His 12

Acid protonates2' OH leaving group

O

O OHP OOO

PyrimidinO5'...

3' phosphate

Second Step:

His 12 is general acid, His 119 is general base


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Geometry of the pentacovalent transition state. The central phosphorus atom is transiently bonded to 5 oxygen atoms. Threeoxygens are coplanar with the phosphorus. The oxygen atoms of theleaving group is at one apex, and the oxygen atom of the attacking groupis at the other apex of the trigonal bipyramid (in-line attack).

Proposed mechanism of RNase A catalysis. The unionized form of His 12 accepts aproton from the 2' OH which enhances its nucleophilicity. The protonated form of His 119begins to donate its proton to the 5' O, and the 2'O begins to form a bond with P to form apentacoordinate transition state. The negative charge that develops is stabilizedelectrostatically by the nearby positively charged side chain of lysine 41. The bond betweenP and the 5'-O breaks when the proton from histidine 119 is completely transferred. At thesame time, a bond between P and the 2'-O becomes fully formed, producing the 2',3'-cyclicintermediate. Hydrolysis of the cyclic intermediate is a reversal of the first stage with H 2Oreplacing the 5'-O component that was removed. Histidine 12 is now the proton donor and

histidine 119 is the proton acceptor.


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Evidence for RNase A mechanism

pH dependence of Vmax/KM for RNase A catalyzedhydrolysis 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 lysineappropriately positioned in the active site. Note hydrogen bonding interactions between cytosine and threonine 45that confer substrate specificity.

Chemical modification . Iodoacetate alkylates histidine 119 or histidine 12 but not both in thesame molecule. Alkylation of either histidine eliminates catalysis. Complex formation withsubstrate or competitive inhibitors protects histidines from modification.


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H O22+Mg 2+Mg OH - + H +

B. Metal ion catalysis 1. Water ionization. A metal ion's charge makes its bound water molecules more acidic thanfree H 2O and therefore a source of OH- ions even below neutral pH (Metal ions have beencalled "Super acids").

2. Charge shielding - metal ions can have charge > +1.3. Oxidation-Reduction

GG

ACA

UCCUGGGCCCCGG

G AA

AG

UAG

UC

AUU G GG5'

5'

Ribozyme

C C

U G U

C AGGAU

Substrate

cleavage site

Hammerhead Catalytic RNA

The Hammerhead Catalytic RNA

The hammerhead ribozyme, like RNase A,catalyzes a transesterification reaction to cleavethe phosphodiester backbone of substrateRNAs yielding products with 5' hydroxyl and2'3'cyclic phosphate termini. Unlike the RNaseA-catalyzed reaction, the hammerhead reaction

does not proceed through hydrolysis of the 2',3'cyclic phosphate.

The hammerhead ribozyme obviously has no amino acid side chains to carry out protontransfer and charge-shielding functions. RNAs are, however, capable of binding metal ionswith 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.


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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 anelectron-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 mobileelectrons (imidazole of His, thiol of Cys, carboxyl of Asp, hydroxyl of Ser).

Chymotrypsin , 25 kd serine protease, catalyzes hydrolysis of proteins in the smallintestine. Chymotrypsin catalyzes hydrolysis of esters as well as peptide bonds which hasbeen useful for analysis of the catalytic mechanism, although not physiologically relevant.

Model reaction in which hydrolysis of acyl enzyme intermediate is slow


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[ p - N

i t r o p

h e n y

l a t e ] , M

Time (min)

10

20

30

2 4 6 8 10 12

chymotrypsin32 M

24 M

16 M

8 M

The plot at left shows the concentration of p -nitrophenol produced a s a function o f time ireactions containing diffe rent concentrationsof chymotrypsin and a large excess of

p-nirophenylacetate. An initial rapid phase("burst") is followed by a slower phase. The

size of the initial burst is proportional to theenzyme concentration. "Burst" kinetic s provide evidence for a stable, enzyme-linkedintermediate.

CCH 3 NO

O

2+ Chymotrypsin

p -Nitrophenylacetate

fast O NO 2-

p -Nitrophenylate

+ CCH 3

O

chymotrypsin

Acyl-enzyme intermediate

slowH O2

H+

CCH 3

O-O

Acetate

+ chymotrypsin

Model reaction in which hydrolysis of acyl-enzyme intermediate is slow

Formation of the acyl-enzyme intermediateoccurs during the initial rapid phase andslower hydrolysis (deacylation) of the acyl-enzyme intermediate occurs during thesecond, slower phase.

o


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First stage in peptide bond hydrolysis: acylation . Hydrolysis of the peptide bond starts with anattack by the oxygen atom of the Ser195 hydroxyl group on the carbonyl carbon atom of thesusceptible bond. The carbon-oxygen bond of this carbonyl group becomes a single bond, andthe 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 facilitatedby Asp102 which ( i) precisely orients the imidazole ring of His57 and ( ii) partly neutralizes thepositive charge that develops on His57 during the transition state. The proton held by theprotonated form of His57 is then donated to the nitrogen atom of the peptide bond that iscleaved. At this stage, the amine component is hydrogen bonded to His57, and the acidcomponent of the substrate is esterified to Ser195. The amine component diffuses away.

Oxyanion hole


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Second stage in peptide hydrolysis: deacylation . The acyl-enzyme intermediateis hydrolyzed by water. Deacylation is essentially the reverse of acylation withwater playing the role as the attacking nucleophile, similar to Ser195 in the firststep. 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, atransient tetrahedral intermediate is formed. His57 then donates a proton to theoxygen atom of Ser195, which then releases the acid component of the substrate,completing the reaction.

Oxyanion hole


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Chymotrypsin catalytic triad Ser195/His57/Asp102 located at the active siteby x-ray crystallography.An important stabilizing feature of the interaction between enzymes and theirsubstrates, is transition state binding. In fact, most enzyme active sites are

organized such that binding to the transition state is preferred over binding toeither substrates or products. The active site of chymotrypsin is arranged tostably interact with the negatively charged carbonyl oxygen of the tetrahedralintermediate this part of the active site is referred to as the oxyanion hole.


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Mechanism of Protein Splicing:

The protein splicing pathwayconsists of four nucleophilicdisplacements. X represents the S orO atom of the Cys/Ser/Thrsidechains.

From: Perler, FB (1998) Cell 92 , 1-4

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Lecture 5a Catalysis