Structural and functional implications of metal ion selection in aminopeptidase P, a metalloprotease with a dinuclear metal center.
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.
Graham
Stephen C
SC
School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia.
Bond
Charles S
CS
Freeman
Hans C
HC
Guss
J Mitchell
JM
D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D008670:Metals; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase