The mechanism of enzyme action.

Abstract

As it is impossible to cover the whole field of enzyme mechanisms in a comparatively brief presentation, it is proposed to illustrate the present state of knowledge by reference in detail to one particular enzyme, namely, bovine pancreatic ribonuclease. There is now an enormous amount of information available about this enzyme, including the complete three-dimensional structure of the protein (Kartha, Bello, and Harker, 1967) and a modified derivative (Richards and Wyckoff, 1968). There is also a great deal of chemical and kinetic information which sheds light on the mechanism of its catalytic action and it is now possible to suggest tentatively the nature of the reaction pathway. The physical basis of the rate enhancement factors, which are of the order of magnitude of 10,10 is still problematical and will not be discussed. The enzyme consists of a single chain of 124 amino acid residues; in general, the molecule is kidneyshaped containing a depression, and there is good reason to believe that the active site is in the depression. Several of the amino acid residues in the region of the active site have been implicated inthecatalytic process. Whilst histidines 12 and 119 are the most important, both lysine 41 (Murdock, Grist, and Hirs, 1966) and aspartate 121 (Anfinsen, 1956) are also essential. Lysine 41 is implicated because the effect of fluorodinitrobenzene, which reacts rapidly with the lysine residue and inactivates the enzyme, is prevented by competitive inhibitors; aspartate 121 is implicated because, whereas removal of the end three amino acids from the C-terminus has no effect on catalytic activity, removal of the next one, ie, aspartate 121, results in complete loss of catalysis. The exact function of these two residues is unknown. By far the most important residues have been shown by experiments with haloacetic acids to be two histidine residues, namely 12 and 119 (see Rabin and Mathias, 1963 for review). Negatively charged alkylating reagents, such as iodoacetic acid and bromoacetic acid, inhibit ribonuclease, but this does not occur with neutral alkylating agents such as iodoacetamide, despite the fact that the latter are generally much more reactive than the former. The reaction of the enzyme with the haloacetic acids is extraordinary, as either one of the two histidines will react with the reagent but never both in the same molecule. Moreover the rate of this reaction is several orders of magnitude greater than that of haloacetic acid with a simple imidazole in aqueous solution. If the rate of alkylation of ribonuclease by iodoacetic acid is measured as a function of pH, a typical bell-shaped curve, resembling an idealized pH profile for enzyme activity, is obtained. The reaction of a simple imidazole with iodoacetic acid does not vary withpH in the same way, but follows a simple titration curve inflecting about thepK of the reacting group. There is obviously an ancillary acid group required for the reaction of the enzyme with iodoacetic acid. As a result of experiments of this sort the concept emerged that in the enzyme these two histidines must be located close together three-dimensionally, in such a way that one of them in the acid form can promote the reactivity of the other towards alkylating reagents. One of the histidines, in the positively charged form, could attract and bind the negative end of the alkylating reagent and juxtapose the reactive carbon atom of the latter to the nitrogen of the other histidine thus promoting its alkylation. Clearly, one imidazole acts as a base and the other as an acid; their pKs are in the region of 6 so that in this pH range there will be an equilibrium mixture of acid and base forms. Which histidine is alkylated would depend amongst other things on the distribution of the charges. This general picture would explain why either of these two histidines, but never both in the same molecule, is alkylated by iodoacetic acid. Competitive inhibitors, which presumably sit on the active site, protect these histidine residues against the action of the alkylating reagents. There is also other evidence which implicates these two histidines in the catalytic activity of the enzyme. That they are indeed not many nanometers apart has been confirmed more recently by x-ray crystallographic studies (Richards and Wyckoff, 1968). The reaction catalysed by ribonuclease is shown in Figure 1. RNA is hydrolysed in two stages 1 coright.