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Keywords:

  • aminopeptidases;
  • wounding;
  • defense response;
  • proteolysis;
  • exopeptidases

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 His6–LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6–LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6–LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6–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 His6–LAP-A enzyme had Km and kcat values similar to the wild-type His6–LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6–LAP-A activity, as most R431 and D347 mutant His6–LAP-As did not hydrolyze dipeptides. The R431K His6–LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. 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.