Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics
Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics
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DOI：
10.1073/pnas.95.12.6636
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发表时间：
1998-06-09
影响因子：
11.1
通讯作者：
Halpert, JR
中科院分区：
文献类型：
--
作者：
Harlow, GR;Halpert, JR
细胞色素P450 3A 4通常被认为是最重要的人类药物代谢酶，并且已知以协同方式催化许多底物的氧化。基于另一个实验室使用各种效应物-底物组合的结构-活性研究以及我们自己使用定点诱变和P450 3A 4的计算机建模的研究，效应物结合的最可能位置是活性位点沿着底物。我们的研究旨在通过分别用较大的Phe和Glu替换残基Leu-211和Asp-214来验证这一假设。预测这些残基构成效应子结合位点的一部分，并且设计取代以通过减小活性位点的大小来模拟效应子的作用。L211 F/D214 E双突变体在低底物浓度下表现出睾酮和孕酮6 β-羟基化速率增加，α-萘醌引起的异向刺激水平降低。双突变体的动力学分析表明，没有同向的协同与任何类固醇底物。在低底物浓度下，野生型酶的类固醇6 β-羟化酶活性被第二类固醇刺激，而L211 F/D214 E显示简单的底物抑制。为了在更机理的水平上分析L211 F/D214 E，进行了光谱结合研究。通过野生型酶的teflazone结合显示同向协同性，而通过L211 F/D214 E的底物结合显示双曲线行为。
Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity, Based on a structure-activity study from another laboratory using various effector-substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6 beta-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by alpha-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6 beta-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were tarried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.