Inactivation of alanine racemase by .beta.-chloro-L-alanine released enzymically from amino acid and peptide C10-esters of deacetylcephalothin

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Abstract
The reactions of a set of amino acid and peptidyl C10-esters of deacetylcephalothin (1-5) have been examined with purified enzymes in vitro. Each of the compounds examined is a substrate for the Escherichia coli TEM-2 .beta.-lactamase, and enzyme-catalyzed hydrolysis of the lactam bond gives release of an amino acid or a peptidyl fragment from a cephem nucleus. 7.beta.-(2-Thienylacetamido)-3-[[(.beta.-chloro-L-alanyl)oxy]methyl]-3-cephem-4-carboxylate (4) gives time-dependent inactivation of E. coli JSR-O alanine racemase in a process that requires .beta.-lactamase for the initial liberation of .beta.-chloro-L-alanine from the cephalosporin. Alanine racemase is similarly inactivated by 7.beta.-(2-thienylacetamido)-3-[[[(.beta.-chloro-L-alanyl)-.beta.-chloro-L-alanyl]oxy]methyl]-3-cephem-4-carboxylate (1), but this inhibition requires the sequential action of both .beta.-lactamase and alanine aminopeptidase. Analysis of the enzymatic transformations of 7.beta.-(2-thienylacetamido)-3-[[[(.beta.-chloro-L-alanyl)-L-alanyl]oxy]methyl]-3-cephem-4-carboxylate (3), monitored by high-field 1H NMR, reveals that (1) .beta.-lactamase releases the dipeptide .beta.-chloro-L-alanyl-L-alanine from 3 and (2) leucine aminopeptidase effects stoichiometric hydrolysis of the dipeptide to .beta.-chloro-L-alanine and L-alanine. These biochemical findings are discussed with reference to the mechanism of antibacterial action of 1 against .beta.-lactamase-producing, penicillin-resistant microorganisms [Mobashery, S., Lerner, S. A., and Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685].