Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae.
In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.
Kull
F
F
Department of Medical Biochemistry, Division of Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Ohlson
E
E
Haeggström
J Z
JZ
D000588:Amines / Q000494:pharmacology; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004851:Epoxide Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011480:Protease Inhibitors / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000588:Amines; D006893:Hydroxyeicosatetraenoic Acids; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012313:RNA; C048698:5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid; D004851:Epoxide Hydrolases; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex; C043134:leukotriene A4 hydrolase