Leukotriene A4 hydrolase: structural and functional properties of the active center.
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that possesses both an epoxide hydrolase activity, i.e., the well-known conversion of LTA4 into the proinflammatory substance LTB4, and a recently discovered peptidase activity. We have employed biochemical/kinetic analyses of native enzyme as well as site directed mutagenesis towards a recombinant enzyme to explore structural and functional properties of the enzyme active center. Thus, we have found that the peptidase activity is selectively stimulated by chloride ions, in a manner that suggests the presence of an anion binding site. Furthermore, a number of mutated enzymes have been constructed, expressed in E. coli, and purified to homogeneity to allow enzyme activity determinations and zinc analyses. The catalytic properties and zinc contents of these mutated enzymes establish the three zinc binding ligands of the protein and identify Glu-296 as a catalytic amino acid, directly involved in the peptidase, but not in the epoxide hydrolase reaction. In conclusion, our data provide strong evidence that the two catalytic activities of LTA4 hydrolase are exerted via non-identical but overlapping active sites.
Haeggström
J Z
JZ
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Wetterholm
A
A
Medina
J F
JF
Samuelsson
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002712:Chlorides / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D001051:Apoenzymes; D002712:Chlorides; D011994:Recombinant Proteins; D003035:Cobalt; D004851:Epoxide Hydrolases; D015032:Zinc; C043134:leukotriene A4 hydrolase