Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250.

Aminopeptidase N (APN; EC 3.4.11.2) purified from Escherichia coli has been crystallized with the optically pure aminophosphinic inhibitor PL250, H(3)N(+)-CH(CH(3))-P(O)(OH)-CH(2)-CH(CH(2)Ph)-CONH-CH(CH(2)Ph)CO(2)(-), which mimics the transition state of the hydrolysis reaction. PL250 inhibits APN with a K(i) of 1.5-2.2 nM and its three-dimensional structure in complex with E. coli APN showed its interaction with the S(1), S'(1) and S'(2) subsites of the catalytic site. In this structure, the Zn ion was shown to be pentacoordinated by His297, His301 and Glu320 of APN and the two O atoms of the phosphinic moiety of PL250. One of these O atoms is also involved in a hydrogen bond to Tyr381, supporting the proposed role of this amino acid in the stabilization of the transition state of the enzymatic process. The strength of the phosphinic zinc binding and the occupancy of the S'(2) subsite account for the 100-fold increase in affinity of PL250 compared with the dipeptide-derived inhibitor bestatin (K(i) = 4.1 x 10(-6) M). Accordingly, the removal of the C-terminal phenylalanine of PL250 resulted in a large decrease in affinity (K(i) = 2.17 x 10(-7) M). Furthermore, it was observed that the C-terminal carboxyl group of the inhibitor makes no direct interactions with the amino acids of the APN active site. Interestingly, PL250 exhibits the same inhibitory potency for E. coli APN and for mammalian enzymes, suggesting that the structure of the complex could be used as a template for the rational design of various human APN inhibitors needed to study the role of this aminopeptidase in various pathologies.