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首页> 外文期刊>Biochemistry >Glu-320 and Asp-323 are determinants of the CYP4A1 hydroxylation regiospecificity and resistance to inactivation by 1-aminobenzotriazole
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Glu-320 and Asp-323 are determinants of the CYP4A1 hydroxylation regiospecificity and resistance to inactivation by 1-aminobenzotriazole

机译：Glu-320和Asp-323是CYP4A1羟基化区域特异性和对1-氨基苯并三唑失活的抗性的决定因素

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摘要

Little information is available on the active site structure of the CYP4A family of enzymes or the mechanism by which their omega-hydroxylation regiospecificity is enforced. We report here that the E320A, D323E, and E320/D323E mutations decrease the catalytic rate of CYP4A1 similar to 5-fold and cause up to a 10-fold shift from omega- to (omega-1)-hydroxylation. The decreased catalytic rate is due to an increase in the uncoupled reduction of molecular oxygen. Tighter binding of 1- and 4-substituted imidazoles to the double mutant than to the other proteins suggests that its active site is less constrained. The reaction of these proteins with phenyldiazene causes heme degradation without the detectable formation of a phenyl-iron complex. CYP4A1 and its E320A mutant are not inactivated by 1-aminobenzotriazole (1-APT), but the D323E and E320A/D323E mutants are inactivated. The resistance of purified CYP4A 1 to inactivation by I-ABT is surprising in view of the fact that 1-ABT causes the loss of the omega-hydroxylase activity both in microsomal preparations and in vivo. Collectively, the results establish that Glu-320, and particularly Asp-323, help to define the active site dimensions, the degree of coupled versus uncoupled turnover, the omega- versus (omega-1)-hydroxylation regiospecificity, and the susceptibility to inactivation by mechanism-based inhibitors. Furthermore, they provide experimental evidence for a structural analogy between the CYP4A1 and P450(BM-3) active sites. [References: 48]
机译：关于CYP4A酶家族的活性位点结构或增强其欧米茄羟化区域特异性的机理的信息很少。我们在这里报告E320A，D323E和E320 / D323E突变降低CYP4A1的催化速率，类似于5倍，并导致从omega-到（omega-1）羟基化多达10倍的转变。降低的催化速率归因于分子氧的未偶联还原的增加。 1-和4-取代的咪唑与双突变体的结合比与其他蛋白质的结合更紧密，表明其活性位点受到的约束较少。这些蛋白质与苯基二氮烯的反应导致血红素降解，而没有可检测到的苯基铁配合物形成。 CYP4A1及其E320A突变体未被1-氨基苯并三唑（1-APT）灭活，但D323E和E320A / D323E突变体被灭活。考虑到1-ABT在微粒体制剂和体内都导致ω-羟化酶活性丧失的事实，纯化的CYP4A 1对I-ABT灭活的抗性是令人惊讶的。总的来说，这些结果表明，Glu-320，尤其是Asp-323，有助于定义活性位点的大小，偶联与非偶联周转的程度，omega-与（omega-1）-羟基化区域特异性以及对失活的敏感性通过基于机制的抑制剂。此外，它们为CYP4A1和P450（BM-3）活性位点之间的结构类比提供了实验证据。 [参考：48]

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