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 boro nates and phosphonates, both of which form covalent tetrahedral adduct s 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 tet rahedral adducts are good inhibitors of serine beta-lactamases, howeve r. 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 P 99. It was found by experiment that the aldehyde, the silanetriol, and the alpha-keto acid (and its methyl ester) of the series were signifi cantly poorer inhibitors than the structurally analogous boronate. Thi s result was explored computationally. From the starting point of the crystal structure of a phosphonate adduct (Lobkovsky et al. Biochemist ry 1994, 33, 6762), the various inhibitors were introduced into the ac tive site and the complete structure relaxed by energy minimization in a force field. Tetrahedral structures derived from analogous substrat es 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 be tween the tetrahedral ligands and the active site was shown to best co rrelate with the electrostatic interactions of the MXY(-) moiety with the two conserved lysine residues of the beta-lactamase active site, h ere Lys 67 and Lys 315. There appeared to be no positive correlation b etween the interaction energy of X(-) with the oxyanion hole and the t otal interaction energy; the oxyanion hole therefore appears to contri bute uniformly to the ligand binding but not to discrimination between ligands. There was, however, a correlation between the active site in teraction energies and the interaction energy between MXY(-) and the H 2(alpha 2) helix dipole. The H2 helix may therefore contribute selecti vely toward catalysis and inhibition. The structures were interpreted in terms of the mechanism of Oefner et al. (Oefner et al. Nature (Lond on) 1990, 343, 284). The relationship of the calculations to the measu red inhibitory properties of the parent molecules is discussed as well as projections to further inhibitor design.