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Abstract: Saccharomyces cerevisiae leukotriene A4 hydrolase (LTA4H) is a bifunctional aminopeptidase/epoxide hydrolase and a member of the M1 family of metallopeptidases. In order to obtain a more thorough understanding of the aminopeptidase activity of the enzyme, two conserved tyrosine residues, Tyr244 and Tyr456, were altered to phenylalanine and the mutant proteins characterized by determining K M and k cat for various amino acid β-naphthylamide substrates. While mutation of Tyr456 exhibited minimal effect on catalysis, mutation of Tyr244 caused an overall 25–100-fold reduction in catalytic activity for all substrates tested. Furthermore, LTA4H Y244F exhibited a 40-fold decrease in affinity for RB-3014, a transition state analog inhibitor, implicating Tyr244 in transition state stabilization. [Copyright &y& Elsevier]

The puromycin-sensitive aminopeptidase (ApPS) is a zinc metallopeptidase involved in the degradation of neuropeptides. Putative catalytic residues of the enzyme, Cys146, Glu338, and Lys396 were mutated, and the resultant mutant enzymes characterized. ApPS C146S exhibited normal catalytic activity, ApPS E338A exhibited decreased substrate binding, and ApPS K396I exhibited decreases in both substrate binding and catalysis. ApPS K396I and ApPS Y394F were analyzed with respect to transition state inhibitor binding. No effect was seen with the K396I mutation, but ApPS Y394F exhibited a 3.3-fold lower affinity for RB-3014, a transition state inhibitor, indicating that Tyr394 is involved in transition state stabilization. [Copyright &y& Elsevier]

Abstract: Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A4 hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues. [ABSTRACT FROM AUTHOR]