Previous studies have shown that β-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 β-lactamases,
however. In this paper, a series of molecules
potentially capable of forming anionic tetrahedral adducts at the
active site
[PhCH2CONHCH2M(XY)-OSer]
have
been assessed as sources of transition-state analogs and as inhibitors
of the class C β-lactamase of Enterobacter
cloacae P99. It was found by experiment that the aldehyde,
the silanetriol, and the α-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 α-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 β-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(α2)
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.