Straub P, Johnson E F, Kemper B
Department of Physiology and Biophysics, University of Illinois at Urbana, Champaign 61801.
Arch Biochem Biophys. 1993 Nov 1;306(2):521-7. doi: 10.1006/abbi.1993.1547.
To determine the requirements for hydrophobic amino acids at position 113 in cytochrome P450 2C2, a series of hydrophobic and uncharged polar amino acids was substituted for isoleucine in P450 2C2 and in C2MstC1, a chimera of P450 2C2 and P450 2C1. Lauric acid hydroxylase activity was determined in COS1 cells transfected with P450 2C2 mutants and both lauric acid and progesterone hydroxylase activities were determined for C2MstC1 variants. In P450 2C2, 40 to 120% of the wild type (omega-1) lauric acid hydroxylase activity was retained in all hydrophobic mutants, but activity was reduced to near background by substitutions of the polar amino acids, tyrosine and cysteine. Likewise, in C2MstC1 mutants, hydrophobic substitutions were 20 to 50% as active as wild type for lauric acid hydroxylation, and polar amino acids again resulted in strong reductions of activity. In contrast, a different pattern of activity for progesterone C21-hydroxylase was observed for C2MstC1 mutants. A valine substitution had a modest effect on activity but substitutions of leucine and alanine reduced progesterone C21-hydroxylase activity 5- to 7-fold, respectively, and the large hydrophobic amino acid, phenylalanine, reduced activity about 30-fold. No changes in the regiospecificity of progesterone hydroxylation were observed for any of the mutants. Similar steady-state levels of immunoprecipitated, radiolabeled protein were observed for each mutant except for the glycine substitution which resulted in degradation of the protein. The different patterns of the effects of the mutations on progesterone and lauric acid hydroxylase activity provide additional support for the critical role of residue 113 in substrate recognition. The low activities in mutant proteins with hydrophilic amino acid substitutions indicate that the hydrophobic nature of this residue is important. The hydrophobic requirements are more stringent for a larger, more rigid steroid substrate than for a saturated fatty acid with a flexible hydrocarbon tail.
为了确定细胞色素P450 2C2中第113位疏水性氨基酸的需求，一系列疏水性和不带电荷的极性氨基酸被替换为P450 2C2和C2MstC1（P450 2C2与P450 2C1的嵌合体）中的异亮氨酸。在用P450 2C2突变体转染的COS1细胞中测定月桂酸羟化酶活性，并且针对C2MstC1变体测定月桂酸和孕酮羟化酶活性。在P450 2C2中，所有疏水性突变体均保留了野生型（ω-1）月桂酸羟化酶活性的40%至120%，但极性氨基酸酪氨酸和半胱氨酸的替换使活性降低至接近背景水平。同样，在C2MstC1突变体中，疏水性替换的月桂酸羟化活性为野生型的20%至50%，极性氨基酸再次导致活性大幅降低。相比之下，观察到C2MstC1突变体的孕酮C21-羟化酶活性模式不同。缬氨酸替换对活性有适度影响，但亮氨酸和丙氨酸的替换分别使孕酮C21-羟化酶活性降低5至7倍，而大的疏水性氨基酸苯丙氨酸使活性降低约30倍。未观察到任何突变体的孕酮羟化区域特异性发生变化。除了甘氨酸替换导致蛋白质降解外，每个突变体的免疫沉淀放射性标记蛋白的稳态水平相似。突变对孕酮和月桂酸羟化酶活性的不同影响模式为第113位残基在底物识别中的关键作用提供了额外支持。亲水性氨基酸替换的突变蛋白活性较低表明该残基的疏水性很重要。对于更大、更刚性的类固醇底物，疏水性需求比对具有柔性烃尾的饱和脂肪酸更为严格。
