Identification of threonine 348 as a residue involved in aminopeptidase A substrate specificity

J Biol Chem. 2009 Apr 17;284(16):10618-26. doi: 10.1074/jbc.M806783200. Epub 2009 Feb 19.

Abstract

Aminopeptidase A (APA; EC 3.4.11.7) is a membrane-bound zinc metalloprotease cleaving in the brain the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the central control of blood pressure in hypertensive animals. We docked the specific APA inhibitor, glutamate phosphonate, in the three-dimensional model of the mouse APA ectodomain in the presence of Ca(2+). In the S1 subsite of this model, the Ca(2+) atom was coordinated with Asp-213, Asp-218,y and Glu-215 and three water molecules, one of which formed a hydrogen bond with the carboxylate side chain of the inhibitor. We report here that the carboxylate side chain of glutamate phosphonate also formed a hydrogen bond with the alcohol side chain of Thr-348. Mutagenic replacement of Thr-348 with an aspartate, tyrosine, or serine residue led to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate GluNA, either in the absence or presence of Ca(2+). In the absence of Ca(2+), the mutations modified the substrate specificity of APA, which was nevertheless restored by the addition of Ca(2+). An analysis of three-dimensional models of the corresponding Thr-348 mutants revealed that the interaction between this residue and the inhibitor was abolished or disturbed, leading to a change in the position of the inhibitor in the active site. These findings demonstrate a key role of Thr-348 in substrate specificity of APA for N-terminal acidic amino acids by insuring the optimal positioning of the substrate during catalysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin III / metabolism
  • Animals
  • Calcium / metabolism
  • Glutamates / chemistry
  • Glutamates / metabolism
  • Glutamyl Aminopeptidase / antagonists & inhibitors
  • Glutamyl Aminopeptidase / chemistry
  • Glutamyl Aminopeptidase / genetics*
  • Glutamyl Aminopeptidase / metabolism*
  • Mice
  • Molecular Conformation
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Organophosphonates / chemistry
  • Organophosphonates / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / metabolism*
  • Substrate Specificity
  • Threonine / chemistry
  • Threonine / metabolism*

Substances

  • Glutamates
  • Organophosphonates
  • Recombinant Proteins
  • glutamate phosphonic acid
  • Angiotensin III
  • Threonine
  • Glutamyl Aminopeptidase
  • Calcium