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Bill Royer Office: LRB 921 Phone: x6-6912. Enzyme Catalysis.

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Bill Royer

Office: LRB 921

Phone: x6-6912

Enzyme Catalysis

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

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.

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‡.

DG‡, 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:

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 DG‡.


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.

If a catalyst lowers the activation barrier by DDG‡, the rate of the reaction is enhanced by the factor eDDG‡/RT. Consequently, a ten-fold rate enhancement requires that DDG‡ = 1.36 kcal/mole, less than the energy of a single hydrogen bond.

(DDG‡ = 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)


k1 >k2

k1 <k2

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


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


















Biologically important electrophiles:


+ C=O





Protons Metal Ions

Carbonyl carbon

Biologically important nucleophilic groups:




Hydroxyl group

+ H+


+ H+

Sulfhydryl group



Amino group



+ H+


Imidazole group

+ H+



Adapted from Voet & Voet, Biochemistry


Ribonuclease A

An example of concerted acid-base catalysis - reaction subject to both general acid and general base catalysis

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 12is general base, His 119is general acid

Second Step:

His 12is general acid,His 119is general base


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.


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.

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.

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").

2. Charge shielding - metal ions can have charge > +1.

3. Oxidation-Reduction


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.


Formation of the acyl-enzyme intermediate occurs during the initial rapid phase and slower hydrolysis (deacylation) of the acyl-enzyme intermediate occurs during the second, slower phase.




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.




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.


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) Cell92, 1-4