Hanioka N, Gonzalez F J, Lindberg N A, Liu G, Gelboin H V, Korzekwa K R
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Biochemistry. 1992 Apr 7;31(13):3364-70. doi: 10.1021/bi00128a009.
Cytochrome P450s CYP2A1 and CYP2A2 exhibit 88% sequence similarity, yet CYP2A1 metabolizes testosterone almost exclusively (90%) at the 7 alpha-position, whereas CYP2A2 forms several metabolites, with 15 alpha-hydroxytestosterone as a major metabolite. One of the regions with relatively low sequence homology corresponds by sequence alignment to the I and J helices of P450cam. Since this region is known to be part of the active site for P450cam, 26 single point and two double point mutants were prepared where the amino acid for one form was substituted with that of the other. Mutant and wild-type enzymes were expressed in Hep G2 cells using the vaccinia virus vector. Analysis of testosterone regioselectivity revealed that 25 of the mutants show the same regioselectivity as the parent wild-type enzymes and three are inactive, suggesting that no single amino acid in this region is totally responsible for the different selectivities of CYP2A1 and CYP2A2. Kinetic analysis of the CYP2A1 mutants showed that four of the mutants with changes near the conserved oxygen-binding region had Km values with much higher and Vmax values much lower than those of the wild-type enzyme and one mutant had a Vmax value twice as high as that of the wild-type enzyme. Deuterium isotope effects on 7 alpha-hydroxxylation were used to determine changes in the rate of reduction and estimate the relative amount of excess water formation. Changes in reduction rates and the amount of water produced are not sufficient to account for the differences in Vmax values, suggesting that the amount of hydrogen peroxide released is a primary determinant for changes in Vmax.
细胞色素P450s CYP2A1和CYP2A2表现出88%的序列相似性，但CYP2A1几乎只（90%）在7α位代谢睾酮，而CYP2A2形成多种代谢产物，其中15α-羟基睾酮是主要代谢产物。序列同源性相对较低的区域之一通过序列比对对应于P450cam的I和J螺旋。由于已知该区域是P450cam活性位点的一部分，制备了26个单点突变体和两个双点突变体，其中一种形式的氨基酸被另一种形式的氨基酸取代。使用痘苗病毒载体在Hep G2细胞中表达突变型和野生型酶。睾酮区域选择性分析表明，25个突变体表现出与亲本野生型酶相同的区域选择性，3个无活性，这表明该区域中没有单个氨基酸完全负责CYP2A1和CYP2A2的不同选择性。CYP2A1突变体的动力学分析表明，在保守的氧结合区域附近发生变化的4个突变体的Km值比野生型酶高得多，Vmax值比野生型酶低得多，1个突变体的Vmax值是野生型酶的两倍。氘同位素对7α-羟基化的影响用于确定还原速率的变化并估计过量水形成的相对量。还原速率和产生的水量的变化不足以解释Vmax值的差异，这表明释放的过氧化氢量是Vmax变化的主要决定因素。
