THE BIFUNCTIONAL ENZYME LEUKOTRIENE-A(4) HYDROLASE IS AN ARGININE AMINOPEPTIDASE OF HIGH-EFFICIENCY AND SPECIFICITY

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino a cid from several tripeptides, typified by arginyl-glycyl-aspartic acid , arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with cat alytic efficiencies (k(cat)/K(m)) greater-than-or-equal-to 1 x 10(6) M -1 s-1. This exceeds by 10-fold the k(cat)/K(m) for its lipid substrat e leukotriene A4. Catalytic efficiency declined for dipeptides which h ad k(cat)/K(m) ratios 10-100-fold lower than tripeptides. Tetrapeptide s and pentapeptides were even poorer substrates with catalytic efficie ncies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripep tide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the pep tide bond cleaved. Peptides with a blocked NH2 terminus were not hydro lyzed. The specificity constant (k(cat)/K(m)) was optimal at pH 7.2 wi th pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albu mins activated the peptidase, increasing tripeptide affinities (K(m)) by 3-10-fold and specificities (k(cat)/K(m)) by 4-13-fold. Two known i nhibitors of arginine peptidases, arphamenine A and B, inhibited hydro lysis of L-arginine p-nitroanilide with dissociation constants = 2.0 a nd 2.5 muM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotrien e A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are ''better'' substrates. This is compatible with a biological role for the peptidase activity of the e nzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accom modates the available data on the interaction of substrates and inhibi tors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide s ubstrates.