Inhibition of chymotrypsin by a complex of ortho-vanadate and benzohydroxamic Acid: structure of the inert complex and its mechanistic interpretation.

The study of enzymes has regularly been informed by the discovery of new inhibitors. With respect to insight into events at the active site of enzymes, related to catalysis, the most informative inhibitors have generally been substrate or product analogues (1), transition state analogues (2–4), or of the mechanism-based variety (5–8). Depending, to a considerable degree, on the nature of the mechanism of catalysis employed by the enzyme concerned and on the class of inhibitor, the final complex may contain the inhibitor either covalently or non-covalently attached to the enzyme. In general, inhibitors that must undergo covalent reaction to achieve the final complex may reach that state by way of transition states that differ in structure, i.e. in geometry and/or charge distribution, from those of the normal enzyme-catalyzed reaction. If such reactions are unusually rapid, however, as would be expected to occur in the case of a particularly effective inhibitor, then the enzyme active site must be able to stabilize the transition state of the reaction leading to inhibition. It follows, therefore, that a new class of inhibitor could be achieved from a stable analogue of this latter transition state. This situation is shown diagrammatically in the free energy/reaction coordinate diagram of Figure 1 where a good transition state analogue of the enzyme-catalyzed reaction (ES‡ analogue) cannot be achieved by a simple non-covalent binding reaction, but only by way of a covalent reaction between E and I that passes through a transition state EI‡, and one that is, in the present example, significantly stabilized by the enzyme (compare the energies of E+I‡ and EI‡). Under these circumstances, EI‡ analogue, a stable analogue of EI‡, and possibly significantly different in structure from ES‡, should also be an effective inhibitor. Figure 1 Serine proteases have been a traditional testing ground for enzyme inhibitors and, in particular, of transition state analogue inhibitors. The central distinguishing feature of an acyl transfer reaction, such as catalyzed by serine proteases, is the anionic tetrahedral intermediate and associated transition states (9). Since serine proteases operate by a double displacement mechanism with a covalent acyl-enzyme intermediate (10), the tetrahedral intermediates, of acylation (1: L denotes a leaving group) and deacylation (2) are covalently bound to the active site serine nucleophile. Transition state analogue inhibitors, therefore, typically take the form of tetrahedral anions covalently bound to the active site serine. Examples are carbonyl adducts, 3, phosphonates, 4, boronates, 5, and arsonates, 6. In each of these instances, crystal structures have shown the inhibitors placed at the active site in a conformation that rationally mimics a tetrahedral intermediate of the enzyme-catalyzed reaction (11). The inhibition of serine proteases by phosphyl derivatives has been studied for many years (12) and, with suitably specific inhibitors, is a very rapid reaction (13–15). It is clear, therefore, that the enzyme active site must actively catalyze this reaction. The transition state of a phosphylation reaction contains penta-coordinated phosphorus and is thought to have a trigonal bipyramidal geometry (16). The enzyme, therefore, must be able to bind and stabilize a species such as 7 and 8, as well as the classical 1–6 (17). From the arguments made above (Figure 1), a stable penta-coordinated structure, bound to the enzyme, should also be an inhibitor. Stable penta-coordinated structures are not common, but among compounds of bio-compatible elements, those of vanadium stand out. Vanadates have long been employed as transition state analogue inhibitors of enzymes catalyzing phosphyl transfer reactions (18–21). Crystal structures of the inhibitory complexes indeed reveal penta-coordinated vanadium (22–25). In view of the above, therefore, we (JHB and RFP) looked for inhibition of α-chymotrypsin by complexes of hydroxamic acids with vanadate, anticipating that complexes of structure 9, analogous to 8, might be formed. We indeed did find inhibition (26), just as we did with another serine protease, elastase, and with a different class of serine hydrolase, the class C β-lactamases (27), but in no case, until now, was the structure of the inert complex determined. In this paper, we report the 1.5 A crystal structure of chymotrypsin in complex with vanadate and benzohydroxamic acid. The structure reveals the inhibitor at the active site and a novel mode of inhibition.