Yanase T, Kagimoto M, Suzuki S, Hashiba K, Simpson E R, Waterman M R
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1989 Oct 25;264(30):18076-82.
Steroid 17 alpha-hydroxylase and 17,20-lyase activities reside within the same polypeptide chain (cytochrome P-450(17 alpha)), and consequently human 17 alpha-hydroxylase deficiencies are characterized by defects in either or both of these activities. Human mutants having these deficiencies represent an excellent source of material for investigation of P-450(17 alpha) structure-function relationships. The CYP17 gene from an individual having partial combined 17 alpha-hydroxylase/17,20-lyase deficiency has been characterized structurally and the homozygous mutation found to be the deletion of the phenylalanine codon (TTC) at either amino acid position 53 or 54 in exon 1. Reconstruction of this mutation into a human P-450(17 alpha) cDNA followed by expression in COS 1 cells led to production of the same amount of immunodetectable P-450(17 alpha) protein as found with expression of the normal human P-450(17 alpha) cDNA. However, 17 alpha-hydroxylase activity of this mutant protein measured in intact cells was less than 37% of that observed upon expression of the wild-type enzyme, whereas 17,20-lyase activity of the mutant was less than 8% of that observed with the normal enzyme. When estimated in intact cells, the Km for 17 alpha-hydroxylation of progesterone was increased by a factor of 2 in the mutant enzyme, whereas the Vmax was reduced by a factor of 3. In order to estimate the kinetic parameters for the 17,20-lyase reaction, microsomes were isolated from transfected COS 1 cells to enrich for this activity. Surprisingly, the specific activity of the mutant 17 alpha-hydroxylase in microsomes was 3-fold less than that observed in intact cells, indicating that the structure of mutant P-450(17 alpha) was dramatically altered upon disruption of COS 1 cells. Apparently the deletion of a single phenylalanine in the N-terminal region of P-450(17 alpha) alters its folding in such a way that both enzymatic activities are dramatically decreased, leading to the partial combined deficiency observed in this individual.
类固醇17α-羟化酶和17,20-裂解酶活性存在于同一条多肽链（细胞色素P-450(17α)）中，因此人类17α-羟化酶缺乏症的特征是这两种活性中的一种或两种存在缺陷。具有这些缺陷的人类突变体是研究P-450(17α)结构-功能关系的极佳材料来源。对一名患有部分联合17α-羟化酶/17,20-裂解酶缺乏症个体的CYP17基因进行了结构表征，发现纯合突变是外显子1中氨基酸位置53或54处苯丙氨酸密码子（TTC）的缺失。将此突变重建到人类P-450(17α) cDNA中，然后在COS 1细胞中表达，产生的免疫可检测P-450(17α)蛋白量与正常人P-450(17α) cDNA表达时相同。然而，在完整细胞中测量的这种突变蛋白的17α-羟化酶活性不到野生型酶表达时观察到的活性的37%，而突变体的17,20-裂解酶活性不到正常酶的8%。在完整细胞中估计时，突变酶中孕酮17α-羟化的Km增加了2倍，而Vmax降低了3倍。为了估计17,20-裂解酶反应的动力学参数，从转染的COS 1细胞中分离出微粒体以富集该活性。令人惊讶的是，微粒体中突变型17α-羟化酶的比活性比在完整细胞中观察到的低3倍，表明突变型P-450(17α)的结构在COS 1细胞破坏后发生了显著改变。显然，P-450(17α) N端区域单个苯丙氨酸的缺失改变了其折叠方式，使得两种酶活性都显著降低，导致该个体出现部分联合缺乏症。
