Enzymes adopt conformations that are structurally and chemically complementary to the transition states of the reactions that they catalyze. Sets of interacting amino acid residues make up sites with the special structural and chemical properties necessary to stabilize the transition state. Enzymes use five basic strategies to form and stabilize the transition state. The first (1) involves the use of binding energy to promote both specificity and catalysis. The others are (2) covalent catalysis, (3) general acid–base catalysis, (4) catalysis by approximation, and (5) metal ion catalysis. The four classes of enzymes examined in this chapter catalyze the addition of water to their substrates but have different requirements for catalytic speed, specificity, and coupling to other processes.

9.1Proteases Facilitate a Fundamentally Difficult Reaction

The cleavage of peptide bonds by chymotrypsin is initiated by the attack by a serine residue on the peptide carbonyl group. The attacking hydroxyl group is activated by interaction with the imidazole group of a histidine residue, which is, in turn, linked to an aspartate residue. This Ser-His-Asp catalytic triad generates a powerful nucleophile. The product of this initial reaction is a covalent intermediate formed by the enzyme and an acyl group derived from the bound substrate. The hydrolysis of this acyl-enzyme intermediate completes the cleavage process. The tetrahedral intermediates for these reactions have a negative charge on the peptide carbonyl oxygen atom. This negative charge is stabilized by interactions with peptide NH groups in a region on the enzyme termed the oxyanion hole.

Other proteases employ the same catalytic strategy. Some of these proteases, such as trypsin and elastase, are homologs of chymotrypsin. Other proteases, such as subtilisin, contain a very similar catalytic triad that has arisen by convergent evolution. Active-site structures that differ from the catalytic triad are present in a number of other classes of proteases. These classes employ a range of catalytic strategies but, in each case, a nucleophile is generated that is sufficiently powerful to attack the peptide carbonyl group. In some enzymes, the nucleophile is derived from a side chain whereas, in others, an activated water molecule attacks the peptide carbonyl directly.

9.2Carbonic Anhydrases Make a Fast Reaction Faster

Carbonic anhydrases catalyze the reaction of water with carbon dioxide to generate carbonic acid. The catalysis can be extremely fast: some carbonic anhydrases hydrate carbon dioxide at rates as high as 1 million times per second. A tightly bound zinc ion is a crucial component of the active sites of these enzymes. Each zinc ion binds a water molecule and promotes its deprotonation to generate a hydroxide ion at neutral pH. This hydroxide ion attacks carbon dioxide to form bicarbonate ion, . Because of the physiological roles of carbon dioxide and bicarbonate ions, speed is of the essence for this enzyme. To overcome limitations imposed by the rate of proton transfer from the zinc-bound water molecule, the most-active carbonic anhydrases have evolved a proton shuttle to transfer protons to a buffer.

282

9.3Restriction Enzymes Catalyze Highly Specific DNA-Cleavage Reactions

A high level of substrate specificity is often the key to biological function. Restriction endonucleases that cleave DNA at specific recognition sequences discriminate between molecules that contain these recognition sequences and those that do not. Within the enzyme–substrate complex, the DNA substrate is distorted in a manner that generates a magnesium ion-binding site between the enzyme and DNA. The magnesium ion binds and activates a water molecule, which attacks the phosphodiester backbone.

Some enzymes discriminate between potential substrates by binding them with different affinities. Others may bind many potential substrates but promote chemical reactions efficiently only on specific molecules. Restriction endonucleases such as EcoRV endonuclease employ the latter mechanism. Only molecules containing the proper recognition sequence are distorted in a manner that allows magnesium ion binding and, hence, catalysis. Restriction enzymes are prevented from acting on the DNA of a host cell by the methylation of key sites within its recognition sequences. The added methyl groups block specific interactions between the enzymes and the DNA such that the distortion necessary for cleavage does not take place.

9.4Myosins Harness Changes in Enzyme Conformation to Couple ATP Hydrolysis to Mechanical Work

Finally, myosins catalyze the hydrolysis of adenosine triphosphate (ATP) to form adenosine diphosphate (ADP) and inorganic phosphate (P_i). The conformations of myosin ATPase domains free of bound nucleotides and with bound ATP are quite similar. Through the use of ADP and vanadate , an excellent mimic of the transition state for ATP hydrolysis bound to the myosin ATPase domain can be produced. The structure of this complex reveals that dramatic conformational changes take place on formation of this species from the ATP complex. These conformational changes are used to drive substantial motions in molecular motors. The rate of ATP hydrolysis by myosin is relatively low and is limited by the rate of product release from the enzyme. The hydrolysis of ATP to ADP and P_i within the enzyme is reversible with an equilibrium constant of approximately 10, compared with an equilibrium constant of 140,000 for these species free in solution. Myosins are examples of P-loop NTPase enzymes, a large collection of protein families that play key roles in a range of biological processes by virtue of the conformational changes that they undergo with various nucleotides bound.

283