A series of mercaptoacetic acid thiol esters have been identified as metallo-beta-lactamase inhibitors. Electrospray mass spectrometry (ESMS) has shown that irreversible inhibition of the Bacillus cereus II metallo-beta-lactamase by SB214751, SB214752, and SB213079 was concomitant with a 90-Da increase in mass of the enzyme. Tryptic digestion of the B. cereus II inhibited with SB214751 illustrated that the peptide fragment, containing the only cysteine of the enzyme, had undergone a mass increment of 90 Da. It was further demonstrated that B. cereus II hydrolyzed this type of compound across the thiol ester bond to yield mercaptoacetic acid. Mercaptoacetic acid is the only molecular fragment common to SB214751, SB214752, and SB213079, and free mercaptoacetic acid does not bind covalently to B. cereus II. Therefore, it is concluded that these compounds inhibit B. cereus II by the mechanism-based delivery of mercaptoacetic acid, forming a disulfide linkage with the active sites cysteine (predicted mass shift = +90 Da) under the aerobic conditions of the assay. The different thiol esters examined had a broad range of potencies against the metallo-beta-lactamases tested. For example SB214751, SB214752, and SB213079 all had 50% inhibitory concentrations of < 10 and > 1,000 microM for the Stenotrophomonas maltophilia L-1 and Bacteroides fragilis CfiA enzymes, respectively. SB216968 was particularly active against the Aeromonas hydrophila CphA metallo-beta-lactamase and was found to be an uncompetitive inhibitor of this enzyme (Ki = 3.9 microM), whereas it exhibited irreversible inhibition of the L-1 enzyme. These observations with this series of compounds have revealed subtle differences between the active sites of different metallo-beta-lactamases. Finally, a novel application for isothermal titration calorimetry for assessing the zinc chelating activity of candidate inhibitors is also presented.

Limited tryptic proteolysis was used to investigate conformational changes of thymidylate synthase from Lactobacillus casei induced by ligand binding. Most of the identified sites of proteolysis were between R72 and R178, a region that includes a large loop containing residues 90-139 that is absent in thymidylate synthase from most other sources. Hydrolysis at both ends of this region was affected by the presence of dUMP. With dUMP, the preference of initial hydrolysis at the N-terminus of this region was switched from R78 to R72, and hydrolysis at R178 was retarded; the latter effect may be primarily a consequence of steric hinderance since R178 is involved in binding the phosphate moiety of dUMP. Orthophosphate had an effect similar to that of dUMP, not only in retarding hydrolysis at the phosphate binding site (R178) but also in retarding hydrolysis at R78 in favor of R72. Alkylation of the catalytically essential sulfhydryl group of thymidylate synthase by iodoacetamide also resulted in R72 being favored over R78 as a site of initial proteolysis. Its effect on hydrolysis at R178 was, as expected, less than that of dUMP or phosphate. These results indicate that dUMP binding induces conformational changes in thymidylate synthase. Phosphate binding and sulfhydryl alkylation also induce conformational changes similar to those resulting from dUMP binding. While the similarity of the proteolytic behavior of thymidylate synthase in the presence of dUMP or phosphate agrees with the report by Finer-Moore et al.(ABSTRACT TRUNCATED AT 250 WORDS)