Aminopeptidase B is structurally related to leukotriene-A4 hydrolase but is not a bifunctional enzyme with epoxide hydrolase activity

Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains 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 for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis. The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity. None of the expressed mutated proteins showed aminopeptidase activity. The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry. From sequence-homology 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 recombinant rat Ap B, both of which had been purified previously [Fukasawa! Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA(4) into LTB4. We then replaced some amino acids in the domain of the rat enzyme similar to the LTA(4)-binding site of LTA(4) hydrolase. However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity. We concluded that Ap B is an MI-family zinc metallopeptidase without epoxide hydrolase activity.