Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A₄ hydrolase.
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.
Seipelt
Rebecca L
RL
Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Bailey
Frank C
FC
Schaible
Alexis
A
Thompson
Michael W
MW
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D001216:Asparagine; D004851:Epoxide Hydrolases; D015032:Zinc; C043134:leukotriene A4 hydrolase