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Graham,a M H Lee,a M J Maher,a W H Simmons,b H C Freemana and J M Gussa
aSchool of Molecular and Microbial Biosciences, University of Sydney, Australia; bDepartment of Molecular and Cellular Biochemistry, Loyola University Chicago, USA
Aminopeptidase P (AMPP) is a proline-specific peptidase that cleaves the N-terminal amino acid residue from a polypeptide chain where the second residue is proline. AMPP is a ubiquitous enzyme that plays an important role in a wide variety of biological processes. Examples include the recycling of cellular proline and, in mammals, the regulation of peptide hormones. Human membrane-bound AMPP is the target of a potential drug, apstatin, which has been shown to regulate blood pressure when used in conjunction with angiotensin-converting enzyme inhibitors. Our previous work on E. coli AMPP has shown that it is a member of the family of pita-bread fold enzymes with a dinuclear metal centre in its active site. Other members of this family include methionine aminopeptidase and prolidase. Structures have now been refined in the presence of inhibitors and in several crystal forms at different pH values. While these previous studies provided a putative mechanism of action for AMPP, they left open a number of questions including the basis of specificity for the N-terminus of the peptide and for proline as the second residue. We have undertaken further structural studies, including determination of the structures of mutant and inhibitor-bound forms of E. coli AMPP, in order to elucidate its mechanism of catalysis. We have shown that, while AMPP and methionine aminopeptidase have similar structures, the chemistry of the secondary amide bond cleaved by AMPP and the nature of the amino acid residues surrounding the active site of AMPP require differences in their mechanisms of catalysis. We have also made a model of the human membrane-bound form of AMPP. This model has permitted us to draw some conclusions about differences in binding affinity of substrates and inhibitors between the E. coli and human membrane-bound forms of the enzyme.