Mode of inhibition of beta-lactamase I and penicillin binding proteins by a novel, synthetic, bifunctional, irreversible monocyclic beta-lactam

{dollar}beta{dollar}-Lactamase I was isolated from a constitutive penicillinase producing strain, Bacillus cereus 569/H. The mode of action of a novel, synthetic, bifunctional, irreversible monocyclic {dollar}beta{dollar}-lactam inhibitor of {dollar}beta{dollar}-lactamase was investigated. The chemical synthesis of this {dollar}beta{dollar}-lactam incorporates anisidine as a spectroscopic probe with absorbance {dollar}>{dollar}300 nm. Structural characterization of the compound was done by IR, MS and NMR. It inhibits the enzyme in a unique mode, namely, it cross-links irreversibly two functional residues in the active site. Chromatographic analysis of the inhibited enzyme showed a retention time of {dollar}sim{dollar}1.5 min longer than the control. The inhibition is irreversible and the stoichiometry of binding to {dollar}beta{dollar}-lactamase is 1:1. The inactivated enzyme is stabilized to denaturation on a reverse phase column. CD spectra of the inhibited, chromatographically purified enzyme showed 42% retention of secondary structure. This was presumptive evidence of cross-linking. Trypsin digestion of the inhibited enzyme gave only one chromophorically labeled peptide. Its FABMS analysis showed it to be a peptide cross-linked by the {dollar}beta{dollar}-lactam residue between Ser-70 and Lys-234. The FABMS data are consistent with there being a gem-diamino function in the cross-linked dipeptide.; Molecular modeling studies reveal that the proposed irreversible cross-linked inhibitor is feasible structurally. Two configurations of the cross-linked inhibitor were modeled, 3R4R and 3S4S, the resulting configurations after inversions at the trans(E) enantiomers. The RR cross-linked model is seen to be the preferred configuration because of the more favorable location of the side chain of the inhibitor.; All classes of {dollar}beta{dollar}-lactamases are irreversibly inhibited by this monocyclic {dollar}beta{dollar}-lactam. No effective {dollar}beta{dollar}-lactam inhibitor has been reported to date for the Zn{dollar}sp{lcub}++{rcub}{dollar} dependent Class B enzyme. This novel inhibitor has provided new avenues for looking into reaction mechanisms of Class B enzymes. It may serve as a probe for the SER-70, LYS-234 motif in other {dollar}beta{dollar}-lactamases.; Penicillin binding proteins (PBPs) are membrane proteins involved in bacterial cell wall biosynthesis. Until now, PBPs have been labeled by radioisotopes for detection by radioautography of their electrophoretograms. A novel, highly sensitive, fluorescence and absorbance-based liquid chromatographic method has been developed in this laboratory for their analysis. This method has several advantages: it is fast and quantitative, cost and time-effective, and avoids increasingly burdensome radioisotope regulatory environment.; The fluorescence/spectroscopic (F/S) probes utilized were a dansylated penicillin, a dansylated cephalosporin, and a dansylated monocyclic {dollar}beta{dollar}-lactam. Competitive binding techniques between novel {dollar}beta{dollar}-lactams generated by our four step synthesis and the probes were used. Reverse phase chromatography showed characteristic PBP binding patterns for each {dollar}beta{dollar}-lactam. Calibrated gel permeation columns were employed for PBP molecular weight determinations. This non-denaturing technique has provided access to multimeric forms of PBPs. Preliminary results yielded by capillary electrophoresis have provided useful MW comparisons with the values reported by SDS-PAGE.