Reciprocal size-effect relationship of the key residues in determining regio- and stereospecificities of DHEA hydroxylase activity in P450 2a5.
Reciprocal size-effect relationship of the key residues in determining regio- and stereospecificities of DHEA hydroxylase activity in P450 2a5.
复制标题
确定 P450 2a5 中 DHEA 羟化酶活性的区域特异性和立体特异性的关键残基的倒数尺寸效应关系。
DOI：
10.1021/bi962654j
复制
发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
Negishi,M
中科院分区：
文献类型：
--
作者：
Uno,T;Mitchell,E;Aida,K;Lambert,MH;Darden,TA;Pedersen,LG;Negishi,M
总的来说，P450 24a4 /2a5系统至少在3个位置（7α， 7β和2α）羟基化脱氢表雄酮。然而，单个P450对其中一种产物表现出高度特异性。通过对野生小鼠和细菌表达的mp450 2a5进行定点诱变，我们将残基117、209和481的功能与每个P450中各自观察到的特异性联系起来。117位的Ala决定7β-羟化酶活性，而该位置的Val决定2α-羟化酶活性。209位的亮氨酸是高脱氢表雄酮7α-羟化酶活性所必需的。用各种疏水氨基酸取代残基481引起了羟基化率的深刻改变，但不影响脱氢表雄酮三个位置的区域和立体特异性。残基481引起的变异也取决于117或209位残基的同一性。结果表明，几个关键残基的大小服从一个协调的互反关系，即p450的底物袋调节以容纳脱氢表雄酮。一项有限的分子模拟研究成功地将脱氢表雄酮结合与一系列关键位置突变体的实验性脱氢表雄酮羟化酶活性联系起来。
Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7α, 7β, and 2α). An individual P450, however, exhibits high specificity to one of these products. Using site-directed mutagenesis ofmP450 2a5 from the wild mouseMusminutoidesand bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7β-hydroxylase activity, whereas Val at this position defines the 2α-hydroxylase activity. Leu at position 209 is essential for high DHEA 7α-hydroxylase activity. The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 also depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.