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Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide 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 peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene 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 enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.

Albumins from several species activated the bifunctional, Zn2+ metalloenzyme amino-peptidase/leukotriene A4 hydrolase (EC 3.3.2.6). Bovine serum albumin, 1 mg/mL, increased hydrolysis of L-proline-p-nitroanilide and leucine-enkephalin by 12-fold and 7-fold, respectively. The apparent Km for L-proline-p-nitroanilide was inversely proportional to the albumin concentration from 0 to 1 mg/mL, declining from 9.4 to 0.7 mM without an appreciable change in apparent Vmax. These data imply a random activation process in which the enzyme-activator complex is catalytically dominant. Hill plots indicated a 1:1 stoichiometric relationship between albumin and enzyme. Secondary plots of slope versus the reciprocal of albumin concentration indicated that it binds to the enzyme with an affinity constant of 0.9 microM. The pH optimum of the nonactivated enzyme occurred at pH 8; the albumin-activated enzyme had an optimum near pH 7. Neither ultrafiltration nor dialysis of albumin altered its activating effect, but boiling abolished it. Albumin did not affect other cytosolic or microsomal leucine aminopeptidases, or gamma-glutamyltransferase. Albumin functions as a nonessential activator, since enzymatic activity was always detectable in its absence. Chloride ions, which activate other Zn2+ metalloenzymes, also activated leukotriene A4 hydrolase/aminopeptidase with an EC50 = 50 mM, increasing its initial velocity 2.2-fold in the absence of albumin. Zn2+ activated the enzyme, increasing its apparent Vmax but not its apparent Km, suggesting it replaced Zn2+ lost from the active site, especially at acidic pH. At concentrations greater than 30-50 microM, Zn2+ was inhibitory. Albumin mitigated the effect of chloride, but not the effect of Zn2+ or that of the competitive inhibitor, captopril.(ABSTRACT TRUNCATED AT 250 WORDS)

Captopril ((2S)-1-(3-mercapto-2-methyl-propionyl)-L-proline) inhibited the bifunctional, Zn(2+)-containing enzyme leukotriene A4 hydrolase/aminopeptidase reversibly and competitively with Ki = 6.0 microM for leukotriene B4 formation and Ki = 60 nM for L-lysine-p-nitroanilide hydrolysis at pH 8. Inhibition was independent of pH between pH 7 and 8, the optimum range for each catalytic activity. Half-maximal inhibition of leukotriene B4 formation by intact erythrocytes and neutrophils required 50 and 88 microM captopril, respectively. In neutrophils and platelets neither 5(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroxyeicosatetraenoic acid, nor leukotriene C4 formation were reduced, indicating selective inhibition of leukotriene A4 hydrolase/aminopeptidase, not 5-lipoxygenase, 12-lipoxygenase, or leukotriene C4 synthase. In whole blood, captopril inhibited leukotriene B4 formation with an accompanying redistribution of substrate toward formation of cysteinyl leukotrienes. The decrease in leukotriene B4 was more substantial than the corresponding increase in cysteinyl leukotrienes suggesting that nonenzymatic hydration predominates over transcellular metabolism of leukotriene A4 by platelets during selective inhibition of leukotriene A4 hydrolase. Enalapril dicarboxylic acid and Glu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro which inhibit angiotensin-converting enzyme: angiotensin I, bradykinin, and N-[3-(2-furyl)acryloyl]Phe-Gly-Gly which are substrates; and chloride ions which activate angiotensin-converting enzyme did not modulate leukotriene A4 hydrolase/aminopeptidase activity. The results indicate that: (i) the sulfhydryl group of captopril is an important determinant for inhibition of leukotriene A4 hydrolase/aminopeptidase, probably by binding to an active site Zn2+; (ii) aminopeptidase and leukotriene A4 hydrolase display differential susceptibility to inhibition; (iii) there is minimal functional similarity between angiotensin-converting enzyme (peptidyl dipeptidase) and leukotriene A4 hydrolase/aminopeptidase; (iv) captopril may be a useful prototype to identify more potent and selective leukotriene A4 hydrolase inhibitors.

Bestatin, an inhibitor of aminopeptidases, was also a potent inhibitor of leukotriene (LT) A4 hydrolase. On isolated enzyme its effects were immediate and reversible with a Ki = 201 +/- 95 mM. With erythrocytes it inhibited LTB4 formation greater than 90% within 10 min; with neutrophils it inhibited LTB4 formation by only 10% during the same period, increasing to 40% in 2 h. Bestatin inhibited LTA4 hydrolase selectively; neither 5-lipoxygenase nor 15-lipoxygenase activity in neutrophil lysates was affected. Purified LTA4 hydrolase exhibited an intrinsic aminopeptidase activity, hydrolyzing L-lysine-p-nitroanilide and L-leucine-beta-naphthylamide with apparent Km = 156 microM and 70 microM and Vmax = 50 and 215 nmol/min/mg, respectively. Both LTA4 and bestatin suppressed the intrinsic aminopeptidase activity of LTA4 hydrolase with apparent Ki values of 5.3 microM and 172 nM, respectively. Other metallohydrolase inhibitors tested did not reduce LTA4 hydrolase/aminopeptidase activity, with one exception; captopril, an inhibitor of angiotensin-converting enzyme, was as effective as bestatin. The results demonstrate a functional resemblance between LTA4 hydrolase and certain metallohydrolases, consistent with a molecular resemblance at their putative Zn2(+)-binding sites. The availability of a reversible, chemically stable inhibitor of LTA4 hydrolase may facilitate investigations on the role of LTB4 in inflammation, particularly the process termed transcellular biosynthesis.