Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato

The importance of two putative Zn2+-binding (Asp347. Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334. Arg336, and Lys262, was evaluated in His(6)-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His(6)-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His(6)-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His,LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His(6)-LAP-A enzyme had K-m and k(cat) values similar to the wild-type His(6)-LAP-A. One catalytic (Arg(431)) and one Zn-binding (Asp(347)) residue were essential for His(6)-LAP-A activity, as most R431 and D347 mutant His(6)-LAP-As did not hydrolyze dipeptides. The R431K His(6)-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in k(cat). Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.