The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity.
The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity.
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发表时间：
1994-04
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通讯作者：
L. Orning;J. Gierse;F. Fitzpatrick
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作者：
L. Orning;J. Gierse;F. Fitzpatrick
白三烯-A4水解酶(EC 3.3.2.6)能从精氨酰甘氨酰-天冬氨酸、精氨酰甘氨酰-甘氨酸和精氨酸组氨基-苯丙氨酸等几种多肽中裂解NH_2末端氨基酸，其催化效率为(kcat/Km)和Gt；或=1×10(6)M~(-1)S~(-1)。这超过了其脂质底物白三烯A4的kcat/Km的10倍。对于kcat/kM比比三肽低10-100倍的二肽，催化效率下降。四肽和五肽是更差的底物，催化效率低于10(3)M-1 S-1。该酶在氨基末端用L-精氨酸优先水解三肽底物和单一氨基酸对硝基苯胺。第二位含有脯氨酸的多肽没有被水解，这表明需要在被切割的多肽键上有一个N-氢。NH2末端封闭的多肽不能被水解。特异性常数(kcat/Km)在pH为7.2时最佳，pK值分别为6.8和7.9，结合能力在pH为8.0时最大。血清白蛋白激活了多肽酶，使三肽亲和力(Km)增加了3-10倍，特异性(Kcat/Km)增加了4-13倍。已知的两种精氨酸肽酶抑制剂Arphamenine A和B能抑制L-精氨酸对硝基苯胺的水解，其解离常数分别为2.0和2.5微米。尽管LTA4水解酶的主要作用被广泛认为是将脂质底物白三烯A4转化为炎症脂质介质白三烯B4，但我们的数据首次表明三肽是更好的底物。这与该酶的多肽酶活性的生物学作用相一致，并可能与该酶在回肠、肝脏、肺和脑等器官中的分布有关。我们提出了一个模型，该模型包含了底物和抑制剂与酶相互作用的现有数据。这个模型可以解释白三烯A4和多肽或对硝基苯胺底物在活性中心的重叠。
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.