Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA.
J Steroid Biochem Mol Biol. 1992 Dec;43(8):1037-43. doi: 10.1016/0960-0760(92)90331-C.
Construction of chimeras and site directed mutagenesis were used to study the regioselectivity and kinetics of testosterone hydroxylation by the cytochrome P450s CYP2A1 and CYP2A2. Although these enzymes exhibit 88% sequence similarity, they catalyze very different regioselective hydroxylations of testosterone. Active chimeras inwhich the first 355 amino acids do not correspond to a single enzyme show broad radioselectivity, whereas the specificity of the parent enzyme is obtained if the first 355 amino acids are unchanged. Therefore, the region between amino acids 275 and 355 is important in maintaining regioselectivity. Single point mutants were constructed for the 13 amino acid differences in this region. For 26 single point and 2 double mutants all active mutants have the same regioselectivity as the parent enzymes. However, kinetic analysis of the CYP2A1 mutants showed that 4 single point mutants and 1 double mutant had kinetic parameters very different from the parent enzyme. All of these substitutions are associated with the conserved dioxygen binding region of the putative I helix predicted from the crystal structure of P450(cam). Deuterium isotope effects were used to determine any changes in the rate of reduction and to estimate the relative amount of excess water formation. Changes in reduction rates are not sufficient to account for the differences in V(max) values. Therefore, it is likely that the amount of hydrogen peroxide formed is a primary determinant of V(max).
利用嵌合体构建和定点突变技术，研究了细胞色素 P450s CYP2A1 和 CYP2A2 对睾酮的羟化反应的区域选择性和动力学。虽然这两种酶具有 88%的序列相似性，但它们催化的睾酮羟化反应具有非常不同的区域选择性。在不对应单一酶的前 355 个氨基酸的活性嵌合体中，表现出广泛的放射选择性，而如果前 355 个氨基酸保持不变，则可以获得母体酶的特异性。因此，氨基酸 275 到 355 之间的区域对于维持区域选择性很重要。在该区域的 13 个氨基酸差异处构建了单点突变体。对于 26 个单点突变体和 2 个双点突变体，所有活性突变体都具有与母体酶相同的区域选择性。然而，对 CYP2A1 突变体的动力学分析表明，有 4 个单点突变体和 1 个双点突变体的动力学参数与母体酶有很大的不同。所有这些取代都与从 P450(cam)晶体结构预测的假定 I 螺旋的保守双氧结合区域相关。氘同位素效应用于确定还原速率的任何变化，并估计过量水形成的相对量。还原速率的变化不足以解释 V(max)值的差异。因此，形成的过氧化氢量可能是 V(max)的主要决定因素。
