Role of THR501 residue in substrate binding and catalytic activity of cytochrome P4501A1.
Role of THR501 residue in substrate binding and catalytic activity of cytochrome P4501A1.
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THR501 残基在细胞色素 P4501A1 的底物结合和催化活性中的作用。
DOI：
10.1006/abbi.2001.2311
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Strobel,HW
中科院分区：
文献类型：
--
作者：
Cvrk,T;Strobel,HW
利用光亲和标记技术，在细胞色素P4501 A1的活性位点上确定了一个氢过氧化枯烯的假定结合区域。Thr 501被确定为最可能的被用作光亲和标记的叠氮枯烯修饰的位点（T. Cvrk和H. W. Strobel，（1998）Arch. Biochem. Biophys. 349，95-104）。为了进一步评估该氨基酸残基的作用，采用定点诱变方法。P4501 A1野生型和两个突变体P4501 A1 Glu 501和P4501 A1 Phe 501在大肠杆菌中表达并纯化，并用于动力学分析以证实Thr 501残基在氢过氧化枯烯结合中的作用。该突变导致在0.5 mM氢过氧化枯烯存在下血红素降解速率降低2 - 4倍。突变不会阻止或显著改变测试底物的结合;然而，2-苯基-2-丙醇（由氢过氧化枯烯产生的产物）与P4501 A1 Glu 501和P4501 A1 Phe 501的结合分别显示出四倍和八倍的降低，表明突变强烈影响氢过氧化枯烯对P4501 A1活性位点的亲和力。枯烯过氧化氢支持的反应的动力学分析表明，这两个突变体表现出增加的Kmax和降低Vmax值为所有测试的基板。此外，突变影响睾酮羟基化的产物分布。通过对P4501 A1 Glu 501和P4501 A1 Phe 501的表征，可以得出Thr 501在过氧化氢异丙苯/P4501 A1相互作用中起重要作用的结论。
A putative binding region for cumene hydroperoxide in the active site of cytochrome P4501A1 was identified using photoaffinity labeling. Thr501 was determined as the most likely site of modification by azidocumene used as the photoaffinity label (T. Cvrk and H. W. Strobel, (1998) Arch. Biochem. Biophys. 349, 95–104). To evaluate further the role of this amino acid residue a site-directed mutagenesis approach was employed. P4501A1 wild type and two mutants, P4501A1Glu501and P4501A1Phe501, were expressed in and purified from Escherichia coli and used for kinetic analysis to confirm the role of Thr501 residue in cumene hydroperoxide binding. The mutation resulted in a two- to fourfold decrease in the rate of heme degradation in the presence of 0.5 mM cumene hydroperoxide. The mutations do not prevent or significantly alter binding of the tested substrates; however, binding of 2-phenyl-2-propanol (product generated from cumene hydroperoxide) to P4501A1Glu501and P4501A1Phe501exhibited four- and eightfold decreases, respectively, suggesting that the mutations strongly affected the affinity of cumene hydroperoxide for the P4501A1 active site. The kinetic analysis of cumene hydroperoxide-supported reactions showed that both mutants exhibit increased Kmand decreased VMaxvalues for all tested substrates. Furthermore, the mutations affected product distribution in testosterone hydroxylation. On the basis of P4501A1Glu501and P4501A1Phe501characterization, it can be concluded that Thr501 plays an important role in cumene hydroperoxide/P4501A1 interaction.