Inhibition studies on the metallo-beta-lactamase L1 from Stenotrophomonas maltophilia

In an effort to identify a competitive inhibitor that can be used in future spectroscopic and crystallographic studies and to better understand the in teraction of a mercaptoacetic acid-thiolester-containing compound with meta llo-beta-lactamase L1 from Stenotrophomonas maltophilia, inhibition studies using two thiol-containing compounds were conducted. N-(2'-Mercaptoethyl)- 2-phenylacetamide is a competitive inhibitor of L1 with a K-i of 50 +/- 3 m u M, and this compound is not a time-dependent inactivator of L1. N-Benzyla cetyl-D-alanylthioacetic acid is a competitive inhibitor of L1 with a K-i o f 1.6 +/- 0.3 mu M. Matrix-assisted laser desorption ionization time-of-fli ght mass spectrometric studies revealed that 2 mol of mercaptoacetate coval ently bind to L1 upon incubation of the enzyme with N-benzylacetyl-D-alanyl thioacetic acid; however, this covalently modified enzyme has the same acti vity as wild-type L1. Last, inhibition studies were used to demonstrate tha t 4-morpholinoethane-sulfonic acid does not inhibit L1, even at concentrati ons up to 300 mM. This work identifies two possible competitive inhibitors which can be used in future structural studies and further demonstrates inh ibitory heterogeneity among the metallo-beta-lactamases. (C) 1999 Academic Press.