Aminopeptidase B is structurally related to leukotriene-A(4) hydrolase butis not a bifunctional enzyme with epoxide hydrolase activity

Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contain s the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases. To determine if these putative zinc-binding residues (His(324), His(328) and Glu(347)) and the active-site Glu(325) essential f or the enzyme activity, we replaced the histidines with tyrosines and the g lutamic acid residues with alanines using site-directed mutagenesis. The cD NAs were expressed in Escherichia coli, and the resulting recombinant prote ins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogen eity. None of the expressed mutated proteins showed aminopeptidase activity . The E325A enzyme contained 1 mol of zinc per mol of protein, and the othe r three mutants, H324Y, H328Y and E347A, did not contain significant amount s of zinc, as determined by atomic absorption spectrometry. From sequence-h omology searches, Ap B is known to be closely related to leukotriene (LT)-A (4) hydrolase (EC 3.3.2.6). We examined human placental Ap B and recombinan t rat Ap B, both of which had been purified previously [Fukasawa! Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determ ine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA(4) into LTB4. We then replaced some amino acids in th e domain of the rat enzyme similar to the LTA(4)-binding site of LTA(4) hyd rolase. However, these mutants, Y408F, N409S and NE409-410SS also did not p ossess any epoxide hydrolase activity. We concluded that Ap B is an MI-fami ly zinc metallopeptidase without epoxide hydrolase activity.