Structural and functional implications of metal ion selection in aminopeptidase P, a metalloprotease with a dinuclear metal center

Biochemistry. 2005 Oct 25;44(42):13820-36. doi: 10.1021/bi0512849.

Abstract

The effect of metal substitution on the activity and structure of the aminopeptidase P (APPro) from Escherichia coli has been investigated. Measurements of activity in the presence of Mn2+, Mg2+, Zn2+, Na+, and Ca2+ show that significant activity is seen only in the Mn-bound form of the enzyme. The addition of Zn2+ to [MnMn(APPro)] is strongly inhibitory. Crystal structures of [MnMn(APPro)], [MgMg(APPro)], [ZnZn(APPro)], [ZnMg(APPro)], [Ca_(APPro)], [Na_(APPro)], and [apo(APPro)] were determined. The structures of [Ca_(APPro)] and [Na_(APPro)] have a single metal atom at their active site. Surprisingly, when a tripeptide substrate (ValProLeu) was soaked into [Na_(APPro)] crystals in the presence of 200 mM Mg2+, the structure had substrate, but no metal, bound at the active site. The structure of apo APPro complexed with ValProLeu shows that the N-terminal amino group of a substrate can be bound at the active site by carboxylate side chains that normally bind the second metal atom, providing a model for substrate binding in a single-metal active enzyme. Structures of [MnMn(APPro)] and [ZnZn(APPro)] complexes of ProLeu, a product inhibitor, in the presence of excess Zn reveal a third metal-binding site, formed by two conserved His residues and the dipeptide inhibitor. A Zn atom bound at such a site would stabilize product binding and enhance inhibition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminopeptidases / chemistry*
  • Aminopeptidases / metabolism*
  • Crystallography, X-Ray
  • Escherichia coli / enzymology
  • Kinetics
  • Metals / metabolism*
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Metals
  • Recombinant Proteins
  • Aminopeptidases
  • X-Pro aminopeptidase