Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species - Comparison with proteinase K and aqualysin I

An extracellular serine proteinase purified from cultures of a psychrotroph ic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47- kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergo es autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares mos t of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. Th e catalytic efficiency (k(cat)/K-m) for the amidase activity of the cold-ad apted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both tow ards heat and denaturants, was found to be significantly lower than of eith er proteinase K or aqualysin I. One or more disulfide bonds in the psychrot rophic proteinase are important for the integrity of the active enzyme stru cture, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aq ualysin I was also partially inactivated by dithiothreitol, but the activit y of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absen ce of a denaturant. The reactivity of the disulfides of the proteins as a f unction of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2-oxidation, as a function of te mperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chain s present in the three related proteinases, and hence a difference in the c ompactness of their protein structures.