Aryl-L-aminoacylamidase activities in extracts of Streptococcus durans.

Two distinct enzymes with aryl-l-aminoacylamidase activity were found in cellular extracts of Streptococcus durans. One of these enzymes was strictly an arylamidase lacking any observable N-terminal exopeptidase activity. The other enzyme functioned as an aminopeptidase capable of catalyzing the hydrolysis of a variety of l-peptide and arylamide substrates. The arylamidase (molecular weight, 80,000) purified 425-fold to homogeneity preferred arylamides containing large hydrophobic side chains, whereas the partially purified aminopeptidase (molecular weight, 300,000) preferred substrates with small nonpolar or basic side chains. Neither enzyme contained any endopeptidase or carboxypeptidase activity. The purified arylamidase was unaffected by metal chelators, but Mn(2+) and Mg(2+) did act as nonessential activators exclusively affecting the maximal velocity. The arylamidase-catalyzed hydrolysis of l-leucyl-p-nitroanilide exhibited a bell-shaped pH dependence for log V(max)/K(m) (pK(1) of 7.2; pK(2) of 8.5), whereas the log V(max)-versus-pH profile showed only an acid limb (pK of 6.8). The ionizable group responsible for the basic limb of the log V(max)/K(m)-versus-pH profile corresponded to the alpha-amino group of the substrate l-leucyl-p-nitroanilide (pK(a) = 8.5). Diazoacetyl-dl-norleucine methyl ester (2 mM) in the presence of 100 mM Cu(2+) caused a rapid inactivation of the enzyme (t((1/2)) of 24 min). Neither parachloromercuribenzoate (0.5 mM) nor N-ethylmaleimide (50 mM) had any effect on the arylamidase activity. Reversible noncompetitive inhibition was observed for iodoacetate (K(i) of 30 mM), N-acetylimidazole (K(i) of 4.0 mM), and ethyl acetimidate (K(i) of 45 mM), although time-dependent irreversible inactivation was not observed with these reagents.