Wu F F, Vergères G, Waskell L
Department of Anesthesia, University of California, San Francisco.
Arch Biochem Biophys. 1994 Feb 1;308(2):380-6. doi: 10.1006/abbi.1994.1054.
In an attempt to understand which amino acids in the membrane anchor of cytochrome b5 might be determinants of its ability to support the cytochrome P450-catalyzed oxidation of selected substrates, the synthetic rat cytochrome b5 gene has been mutated by site-directed mutagenesis. The mutant proteins have been expressed in Saccharomyces cerevisiae, purified and assayed for their ability to support the cytochrome P450-catalyzed metabolism of the cytochrome b5 requiring substrate methoxyflurane (G. Vergères and L. Waskell, 1992, J. Biol. Chem. 267, 12583-12591). The rate of reduction of the cytochromes b5 by cytochrome P450 reductase has been examined by stopped-flow spectrophotometry to determine whether an altered rate of reduction of cytochrome b5 could explain the observed activity of cytochrome b5 in the purified reconstituted mixed-function oxidase system. A mutant in which the 22-amino-acid membrane anchor was replaced by a sequence of 22 leucines was unable to support methoxyflurane metabolism in the reconstituted system and was reduced by cytochrome P450 reductase at a rate (k = 4.5 x 10(-3) s-1) slow enough to explain this finding. Comparison of the rate of reduction of this mutant cytochrome b5 in 0.025% Tergitol and 40 microM dilauroylphosphatidylcholine suggests that this slow rate of reduction may be explained partially by aggregation of the polyleucine protein. The Pro115Stop mutant protein, which has been truncated by 19 amino acids in its COOH terminus resulting in a protein with one-half of the putative membrane anchor, supports methoxyflurane oxidation at 12-20% of the rate of the wild type protein. In addition it is reduced by cytochrome P450 reductase at a rate which should be capable of supporting a normal rate of production formation. The fact that the Pro115Stop mutant can be reduced at a rate capable of supporting a normal rate of methoxyflurane oxidation but in fact only supports methoxyflurane oxidation at 30% of the normal rate suggests that the mutant protein is deficient in its interactions with cytochrome P450. The mutant proteins, Pro115Ala and Ala116Pro, behaved essentially as did the wild type protein demonstrating that the presence (Pro115Ala) or absence (Ala116Pro) of an alpha helix in the middle of the putative membrane-binding domain of cytochrome b5 was not a determinant of the interaction of cytochrome b5 with cytochrome P450 reductase and cytochrome P450.(ABSTRACT TRUNCATED AT 400 WORDS)
为了弄清楚细胞色素b5膜锚定区中的哪些氨基酸可能是其支持细胞色素P450催化特定底物氧化能力的决定因素，已通过定点诱变对合成的大鼠细胞色素b5基因进行了突变。突变蛋白已在酿酒酵母中表达、纯化，并检测了它们支持细胞色素P450催化细胞色素b5所需底物甲氧氟烷代谢的能力（G. Vergères和L. Waskell，1992年，《生物化学杂志》267卷，12583 - 12591页）。已通过停流分光光度法检测细胞色素P450还原酶还原细胞色素b5的速率，以确定细胞色素b5还原速率的改变是否能解释在纯化的重组混合功能氧化酶系统中观察到的细胞色素b5活性。一个突变体，其22个氨基酸的膜锚定区被22个亮氨酸序列取代，在重组系统中无法支持甲氧氟烷代谢，并且被细胞色素P450还原酶还原的速率（k = 4.5×10⁻³ s⁻¹）足够慢，足以解释这一发现。在0.025%的吐温 - 100和40微摩尔二月桂酰磷脂酰胆碱中对该突变细胞色素b5还原速率的比较表明，这种缓慢的还原速率可能部分是由聚亮氨酸蛋白的聚集所导致的。Pro115Stop突变蛋白在其COOH末端被截短了19个氨基酸，产生了一个具有假定膜锚定区一半的蛋白，其支持甲氧氟烷氧化的速率为野生型蛋白的12 - 20%。此外，它被细胞色素P450还原酶还原的速率应该能够支持正常的产物形成速率。Pro115Stop突变体能够以支持正常甲氧氟烷氧化速率的速率被还原，但实际上仅以正常速率的30%支持甲氧氟烷氧化，这一事实表明该突变蛋白在与细胞色素P450的相互作用方面存在缺陷。突变蛋白Pro115Ala和Ala116Pro的行为与野生型蛋白基本相同，表明细胞色素b5假定膜结合结构域中间α螺旋的存在（Pro115Ala）或缺失（Ala116Pro）不是细胞色素b5与细胞色素P450还原酶和细胞色素P450相互作用的决定因素。（摘要截短至400字）
