Contribution of molecular modeling and site-directed mutagenesis to the identification of a new residue, glutamate 215, involved in the exopeptidase specificity of aminopeptidase A

Biochemistry. 2003 Dec 23;42(50):14785-93. doi: 10.1021/bi034358u.


Aminopeptidase A is a zinc metalloenzyme that generates brain angiotensin III, which exerts a tonic stimulatory action on blood pressure in hypertensive animals. We have previously constructed a three-dimensional model of the ectodomain of this enzyme, using the crystal structure of leukotriene A4 hydrolase/aminopeptidase as a template. According to this model, Glu-215, which is located in the active site, hydrogen bonds to the amino moiety of the inhibitor, 4-amino-4-phosphonobutyric acid (GluPhos), a phosphonic acid anologue of glutamic acid. Replacement of this residue with an aspartate or an alanine in the model abolished this interaction and led to a change in the position of the inhibitor in the active site. Mutagenic replacement of Glu-215 with an aspartate or an alanine drastically reduced the affinity of the recombinant enzymes for the substrate by a factor of 10 or 17, respectively, and the rate of hydrolysis by a factor of 14 or 6, respectively. Two isomers of GluPhos with different N-terminal amine positions differed considerably in their ability to inhibit the wild type (by a factor of 40), but not the mutated enzymes. These results, together with the interaction predicted by the model, demonstrate that Glu-215 interacts with the N-terminal amine of the substrate, thereby contributing, together with Glu-352, to the determination of the exopeptidase specificity of aminopeptidase A.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cricetinae
  • Enzyme Activation / genetics
  • Exopeptidases / chemistry*
  • Exopeptidases / genetics
  • Glutamates / genetics
  • Glutamic Acid / chemistry*
  • Glutamic Acid / genetics
  • Glutamyl Aminopeptidase / antagonists & inhibitors
  • Glutamyl Aminopeptidase / chemistry*
  • Glutamyl Aminopeptidase / genetics*
  • Histidine / genetics
  • Mice
  • Models, Molecular*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Organophosphonates
  • Protein Structure, Tertiary / genetics
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemical synthesis
  • Recombinant Proteins / isolation & purification
  • Sequence Alignment
  • Substrate Specificity / genetics
  • Transfection


  • Glutamates
  • Organophosphonates
  • Recombinant Proteins
  • glutamate phosphonic acid
  • glutamate thiol
  • Glutamic Acid
  • Histidine
  • Exopeptidases
  • Glutamyl Aminopeptidase