EFFECT OF VARIOUS COMPOUNDS ON ENKEPHALIN HYDROLYSIS BY AN AMINOPEPTIDASE FROM THE THERMOPHILES THERMOMONOSPORA FUSCA ATCC-27730 AND THERMUS THERMOPHILUS ATCC-27634

  • 1 January 1983
    • journal article
    • research article
    • Vol. 41  (1) , 149-155
Abstract
The microbial peptides amastatin and bestatin, as well as several dipeptide analogs 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 Tyr-Gly bond of Leu- and Met-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, tetra-peptides and larger molecules containing the Met-enkephalin sequence. The Tf enzyme has a pH optima of 7.5, Km of 667 .mu.M and Vmax of 92 nmol/min per mg of protein; the Tt enzyme, with a pH optimum of 7.2, has a Km of 400 .mu.M and Vmax of 33 nmol/min per 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 Co2+, Mg2+ and Mn2+; ions with an inhibitory effect were Al3+, Ca2+, Cu2+, Hg2+ and Zn2+. The enzymes are not glycoproteins since they pass unretained through a Con A-Sepharose column.