The biochemical and molecular characterization of recombinant Bacillus subtilis tripeptidase (PepT) as a zinc-dependent metalloenzyme

Aminopeptidases catalyze the release of N-terminal Introduction amino acid residue from polypeptides and peptides, and most of them are known to be me talloenzymes, A tripeptidase gene (pepT) of Bacillus subtilis was expressed in Escherichia coli, and the resulting recombinant PepT was purified in an active form through sequential chromatographies. The addition of Zn2+ or C o2+ increased the enzymatic activity by approximately two fold. The points at which Zn2+ and Co2+ Stimulated a half-maximum activity of the PepT were 650nM and 1,700nM, respectively. The measurement of the metal content showe d that this enzyme contained 0.26 atom of Zn2+ per molecule with essentiall y the absence of Co2+ and others, and 0.53 atom of Zn2+ with 1.5-fold incre ase of activity when reconstituted with Zn2+, Consistent with this result, this enzyme is much readily refolded in the presence of Zn2+ than Co2+. To further delineate the structure and function relations, we made serial dele tion mutants and analyzed their enzymatic activities. Of eight deletion mut ants, only a mutant lacking the N-terminal 66 amino acid residues retained enzymatic activity. The mutant enzyme, however, required a concentration of Zn2+ ion at least ten-fold higher to reach maximum activity without signif icantly affecting kinetic parameters such as K-m and V-max compared to the full length PepT. Taken together, these data suggest that the B. subtilis P epT is likely to be a Zn2+-dependent metalloenzyme and that the N-terminal region of the PepT stabilizes Zn2+-binding.