Effectiveness of tetrahedral adducts as transition-state analogs and inhibitors of the class C beta-lactamase of Enterobacter cloacae P99.

Previous studies have shown that beta-lactamases are inhibited by boronates and phosphonates, both of which form covalent tetrahedral adducts with the active-site serine residue. These have been interpreted, as have similar complexes formed with serine proteinases, as transition-state analog structures. Not all molecules capable of forming such tetrahedral adducts are good inhibitors of serine beta-lactamases, however. In this paper, a series of molecules potentially capable of forming anionic tetrahedral adducts at the active site [PhCH(2)CONHCH(2)M(XY)(-)OSer] have been assessed as sources of transition-state analogs and as inhibitors of the class C beta-lactamase of Enterobacter cloacae P99. It was found by experiment that the aldehyde, the silanetriol, and the alpha-keto acid (and its methyl ester) of the series were significantly poorer inhibitors than the structurally analogous boronate. This result was explored computationally. From the starting point of the crystal structure of a phosphonate adduct (Lobkovsky et al. Biochemistry 1994, 33, 6762), the various inhibitors were introduced into the active site and the complete structure relaxed by energy minimization in a force field. Tetrahedral structures derived from analogous substrates were similarly treated, and the final structures were analyzed both structurally and energetically. From the point of view of energy, it was found that the boronate (M = B, X = Y = OH), phosphonate (M = P, X = Y = O), and carbon substrate-derived analog (M = C, X = O, Y = OH) interacted comparably strongly in a noncovalent sense with the active-site residues, while the aldehyde (M = C, X = O, Y = H), silicate (M = Si, X = O, Y = OH), and alpha-keto acid derivatives (M = C, X = O, Y = COR) interacted more weakly. The order of energies of interaction between the tetrahedral ligands and the active site was shown to best correlate with the electrostatic interactions of the MXY(-) moiety with the two conserved lysine residues of the beta-lactamase active site, here Lys 67 and Lys 315. There appeared to be no positive correlation between the interaction energy of X(-) with the oxyanion hole and the total interaction energy; the oxyanion hole therefore appears to contribute uniformly to the ligand binding but not to discrimination between ligands. There was, however, a correlation between the active site interaction energies and the interaction energy between MXY(-) and the H2(alpha 2) helix dipole. The H2 helix may therefore contribute selectively toward catalysis and inhibition. The structures were interpreted in terms of the mechanism of Oefner et al. (Oefner et al. Nature (London) 1990, 343, 284). The relationship of the calculations to the measured inhibitory properties of the parent molecules is discussed as well as projections to further inhibitor design.