Effect of various compounds on enkephalin hydrolysis by an aminopeptidase from the thermophiles Thermomonospora fusca ATCC 27730 and Thermus thermophilus ATCC 27634.
The microbial peptides amastatin and bestatin as well as several dipeptide analogues of the latter exerted little or no inhibitory effect on enkephalin hydrolysis by an aminopeptidase purified from the thermophiles Thermomonospora fusca, ATCC 27730 (Tf) and Thermus thermophilus, ATCC 27634 (Tt). The enzyme catalyzes the cleavage of the tyrosyl-glycyl bond of leucine- and methionine-enkephalin. Intermediate compounds having the same amino acid sequence as the parent substrate disclosed that the residual tetrapeptide can be further degraded to its constituent parts. Each preparation also hydrolyzes to varying extents neutral dipeptides, tripeptides, tetrapeptides, can be further degraded to its constituent parts. Each preparation also hydrolyzes to varying extents neutral dipeptides, tripeptides, tetrapeptides, and larger molecules containing the Met-enkephalin sequence. The Tf enzyme has a pH optimum of 7.5, Km of 667 microM and Vmax of 92 nmol/min/mg of protein; the Tt enzyme, with a pH optimum of 7.2 has a Km of 400 microM and Vmax of 33 nmol/min/mg of protein. Activated by dithiothreitol (DTT) and inactivated by p-chloro- and p-hydroxymercuribenzoate, both are sulfhydryl enzymes. The activity lost by hydrolysis against EDTA can be restored, wholly or in part, by Co+2, Mg+2, and Mn+2; ions with an inhibitory effect were A1+3, Cd+2, Cu+2, Hg+2, and Zn+2. The enzymes are not glycoproteins since they pass unretained through a Con A-Sepharose column.
D000192:Actinomycetales / Q000201:enzymology*; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004745:Enkephalins / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013824:Thermus / Q000201:enzymology*
D004745:Enkephalins; D008670:Metals; D013439:Sulfhydryl Reagents; D000626:Aminopeptidases