Effect of bestatin analogues and other compounds on enkephalin hydrolysis by an aminopeptidase from the mesophiles pseudomonas sp ATCC 11299A and chromobacterium violaceum ATCC 12540.
In our studies on newly synthesized compounds for their potential analgesic effect, we decided for purposes of convenience and economy to investigate non-mammalian sources for the presence of enkephalin degrading enzymes. An aminopeptidase that catalyzes the hydrolysis of the tyrosylglycyl bond of leucine- and methionine enkephalin was purified from the mesophiles Pseudomonas sp ATCC 11299a (Ps) and Chromobacterium violaceum ATCC 12540 (Cv). Each preparation also hydrolyzed to varying extents neutral dipeptides, tripeptides, tetrapeptides and amino acid beta-naphthylamides. The Ps enzyme has a pH optimum of 6.8, Km of 80 microM and a Vmax of 6.7 nmoles/min/mg of protein. The Cv enzyme has a pH optimum of 6.8-7.2, Km of 111 microM and a Vmax of 42 nmoles/min/mg of protein. Both are sulfhydryl enzymes since they are activated by dithiothreitol (DTT) and inactivated by p-chloro- and p-hydroxymercuribenzoate. They are not glycoproteins since they pass unretained through a Con A-Sepharose column. The activity lost by dialysis against EDTA can be restored, wholly or in part, by Co+2, Mg+2, Mn+2 and Ni+2; ions exerting an inhibitory effect were A1+3, Cd+2, Cu+2, Hg+2 and Zn+2. From a range of organic compounds, the greatest inhibition was elicited by the microbial peptides amastatin and bestatin. Several dipeptide analogues of bestatin, synthesized from DL-threo-2-amino-3-hydroxy-3-phenylpropanoic acid (AHPP) as the N-terminal residue in order to define the stereospecific requirements of the alpha, beta-functional groups for maximal activity, were not as active as the parent compound.
D000626:Aminopeptidases / Q000378:metabolism*; D002861:Chromobacterium / Q000201:enzymology*; D004151:Dipeptides / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D004151:Dipeptides; D013439:Sulfhydryl Reagents; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme; D007930:Leucine; C012211:ubenimex