Orning L, Gierse J K, Fitzpatrick F A
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
J Biol Chem. 1994 Apr 15;269(15):11269-73.
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.
白三烯 - A4水解酶（EC 3.3.2.6）能从几种三肽中切割出氨基末端氨基酸，以精氨酰 - 甘氨酰 - 天冬氨酸、精氨酰 - 甘氨酰 - 甘氨酸和精氨酰 - 组氨酰 - 苯丙氨酸为典型代表，其催化效率（kcat/Km）≥1×10⁶ M⁻¹ s⁻¹。这比其脂质底物白三烯A4的kcat/Km高出10倍。二肽的催化效率下降，其kcat/Km比值比三肽低10 - 100倍。四肽和五肽是更差的底物，催化效率低于10³ M⁻¹ s⁻¹。该酶优先水解氨基末端带有L - 精氨酸的三肽底物和单个氨基酸对硝基苯胺。第二位带有脯氨酸的肽不被水解，这表明在切割的肽键处需要一个N - 氢。氨基末端被封闭的肽不被水解。特异性常数（kcat/Km）在pH 7.2时最佳，pK值分别为6.8和7.9；结合在pH 8.0时最大。血清白蛋白激活该肽酶，使三肽亲和力（Km）增加3 - 10倍，特异性（kcat/Km）增加4 - 13倍。两种已知的精氨酸肽酶抑制剂，阿法美宁A和B，抑制L - 精氨酸对硝基苯胺的水解，解离常数分别为2.0和2.5 μM。尽管白三烯A4水解酶的主要作用被广泛认为是将脂质底物白三烯A4转化为炎性脂质介质白三烯B4，但我们的数据首次表明三肽是“更好的”底物。这与该酶的肽酶活性的生物学作用相符，并且可能与该酶在回肠、肝脏、肺和脑等器官中的分布有关。我们提出了一个模型，该模型容纳了关于底物和抑制剂与该酶相互作用的现有数据。这个模型可以解释白三烯A4与肽或对硝基苯胺底物水解活性位点的重叠。
