PURIFICATION AND CHARACTERIZATION OF INTRACELLULAR AMINOPEPTIDASE FROM PSEUDOMONAS-FLUORESCENS ATCC-948

Pseudomonas fluorescens ATCC 948 expressed cell-associated peptidase activity, which was shown by subcellular fractionation to be primarily in the cytoplasmic fraction. An aminopeptidase of broad specificity was purified 129-fold over the crude extract with an 11% recovery by ion-exchange chromatography, biogel filtration, and affinity chromatography. The enzyme had a monomeric structure and a molecular weight of about 50,000. The optimal activity occurred at pH 7.5 and 45-degrees-C. A heat treatment of 75-degrees-C for 1 min was necessary to inactivate the enzyme irreversibly. The aminopeptidase was strongly activated by Co2+, completely inhibited by EDTA and 1,10-phenanthroline, and weakly inhibited by sulfhydryl-blocking agent, suggesting that the aminopeptidase likely is a metalloenzyme with a thiol group at its active site. The enzyme showed high activity with beta-napthylamide derivatives that had a hydrophobic AA (Leu, Ala, or Phe) or diaminomonocarboxylic acid (Lys or Arg) at the N terminus. The occurrence of only one aminopeptidase active on Leu, Lys, or Arg aminoacyl bonds was demonstrated by the competitive inhibition of Lys-beta-naphthylamide and Arg-beta-naphthylamide on the activity against Leu-p-nitroanilide. Kinetic studies conducted on Leu-beta-naphthylamide showed a Michaelis-Menton constant of .3 mM, a maximum velocity of 10 nkat, and an energy of activation of 6200 cal/mol.