Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V(V)), the nature of these complexes at neutral pH and submillimolar concentrations, the conditions under which such complexes inhibit various serine amidohydrolases, is not well established. A series of qualitative and quantitative experiments, involving UV/vis, (1)H NMR, and (51)V NMR spectroscopies, established that both 1:1 and 1:2 vanadate/hydroxamate complexes form at pH 7.5, with the former dominating at submillimolar concentrations. Formation constants for the complexes of several aryl and alkyl hydroxamic acids were determined; for example, for benzohydroxamic acid, the stepwise formation constants of the 1:1 and 1:2 complexes were 3000 and 400 M(-1), respectively. The (51)V chemical shift of the 1:1 4-nitrobenzohydroxamic acid complex was -497 ppm, and that of its unsubstituted analogue was -498 ppm. A (1)H-(15)N HSQC spectrum of the 4-nitrobenzo-(15)N-hydroxamic acid/vanadate complex indicated the presence of an N-H group with (15)N and (1)H chemical shifts of 115 and 5.83 ppm, respectively. A (13)C NMR spectrum of the complex of 4-nitrobenzo-(13)C-hydroxamic acid with vanadate displayed a resonance at 170.1 ppm and thus a coordination-induced shift (CIS) of +3.8 ppm. In contrast, the CIS value of an established 1:2 complex, thought to contain chelated hydroxamic acid ligands, was +11.9 ppm. These spectral data led to the following structural picture of 1:1 complexes of vanadate and aryl hydroxamic acids. They contain penta- or hexa-coordinated vanadium. The ligand is in the hydroxamate rather than hydroximate form. The ligand is presumably bound to vanadium through the hydroxamic hydroxyl oxygen, but the hydroxamic acid carbonyl oxygen interacts weakly with vanadium. These species are the most likely candidates for the inhibitors of serine amidohydrolases found in vanadate/hydroxamic acid mixtures.

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.

Serine proteases, like serine beta-lactamases, are rapidly and covalently inhibited by suitably designed phosph(on)ates. The active sites of these enzymes must, therefore, be able to stabilize the pentacoordinated transition states of phosphyl transfer reactions as well as the tetrahedral transition states of acyl transfers. It follows that these enzymes should also be inhibited by molecules capable of generating inert pentacoordinated species. We (J.H.B. and R.F.P.) have previously shown that these enzymes are, in fact, rapidly and reversibly inhibited by 1:1 complexes of vanadate and hydroxamic acids. In this paper, we present the first crystal structure of an acyl transferase inhibited by vanadate. The complex of vanadate and benzohydroxamic acid is a competitive inhibitor of alpha-chymotrypsin with a KI value of 16 muM. In the structure, obtained at a resolution of 1.5 A, the protein is conformationally little different from the apoenzyme. The vanadium, in a distorted octahedral ligand field, is covalently bound to the active site serine oxygen group. One oxgen ligand, presumably anionic, is located in the oxyanion hole. Another is directed roughly in the direction of the acyl transfer leaving group, and a third in the direction of the S2 site. The hydroxamate is bound to vanadium through the hydroxyl oxygen and also, more weakly, through the carbonyl group, to form a five-membered chelate ring. The effect of this chelation is to place the phenyl group of the inhibitor into the important S1 specificity site. The hydroxamate oxygen is directed in line away from the Ser195 Ogamma, approximating the direction of departure of a leaving group in phosphyl transfer. The entire complex can be seen as a reasonable mimic of a phosphyl transfer transition state where the leaving group is extended into the S1 site.

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