Modified amino acids and peptides as potential inhibitors of bacterial cell wall biosynthesis

In this work we have synthesized a number of modified amino acids and peptides which were supposed to be analogues of the substrate, the postulated intermediate and the product of the reaction catalysed by D-alanine:D-alanine ligase, a key enzyme in the D-alanine branch of bacterial cell wall peptidoglycan assembly. Most of these were crystalline solids, and were prepared as both L and D isomers. When these compounds were subjected to their biological activity it was found that they were unlikely to be inhibitors of the bacterial cell wall biosynthesis. The reduced dipeptide L-alanyl-(CH2-NH)-L-alanine was able to inhibit the growth of S.aureus 6571 in solid as well as liquid media, and its inhibitory effect was stopped in the presence of the dipeptide L-alanyl-L-alanine and the tripeptide L-alanyl-L-alanyl-L-alanine but not by L-alanine. The reduced dipeptide was suggested to act intracellularly as a whole molecule. The N-protected bromo and chloromethyl ketones of alanine were also found to inhibit the growth of S.aureus 6571 and E.coli W3110 in the solid and liquid media, and these were suggested to inhibit a proteinase inside the bacterium. The enzyme D-alanine: D-alanine ligase was purified from E.faecalis (NCIB) 6459) to a specific activity of 8.194 μmol min^-1mg^-1 of protein and its K_m and V_max values were determined. The ligase was also purified from E.coli M72 (pDOC87) to a specific activity of 18.9 μmol min-1mg-1 of protein. E.coli ligase was found to have a Mr of 31,000 ± 1000 by SDS polyacrylamide gel electrophoresis. The N-terminal sequence of ten residues of the ligase was found to be consistent with that predicted from the complete gene sequence reported by Robinson et al. (1986). Finally, the potency of the synthetic inhibitors on E.coli D-alanine: D-alanine ligase was studied in vitro. The intermediate analogues D-(2-amino-1-hydroxypropyl) phosphonate and D-(2-aminopropyl) phosphate were more inhibitory than the substrate analogues D-alanine chloromethyl ketone, D-alanine bromomethyl ketone and 4-amino-3-hydroxy pentanoic acid, and the product analogue D-alanyl-(CH_2-NH)-D-alanine. The kinetics of the ligase inhibition by D-(2-amino-1-hydroxylpropyl) phosphonate and D-(2-aminopropyl) phosphate were studied, and the kinetic constants are summarized. The inhibition of E.coli ligase by the aminoalkyl phosphinate of Merck was also investigated. This compound was found to be a parabolic competitive inhibitor and the kinetic constants are given. ATP was shown not to be required for the ligase inhibition by this compound, and no time-dependent process was obtained. It is suggested that the aminoalkyl phosphinate could be a classical inhibitor of E.coli ligase.