Matsuura K, Deyashiki Y, Sato K, Ishida N, Miwa G, Hara A
Biochemistry Laboratory, Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu 502, Japan.
Biochem J. 1997 Apr 1;323 ( Pt 1)(Pt 1):61-4. doi: 10.1042/bj3230061.
Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors.
人肝脏二氢二醇脱氢酶同工酶（DD1和DD2）仅七个氨基酸残基被取代，它们在甾体底物特异性和抑制剂敏感性方面存在显著差异：DD1具有20α-羟基类固醇脱氢酶活性，对1,10-菲咯啉敏感，而DD2氧化3α-羟基类固醇，并被胆汁酸高度抑制。在本研究中，我们将DD1的七个残基（苏氨酸-38、精氨酸-47、亮氨酸-54、半胱氨酸-87、缬氨酸-151、精氨酸-170和谷氨酰胺-172）定点突变为DD2的相应残基（分别为缬氨酸、组氨酸、缬氨酸、丝氨酸、甲硫氨酸、组氨酸和亮氨酸）。在这七个突变中，只有将亮氨酸-54替换为缬氨酸产生了一种与DD2几乎具有相同性质的酶。在其他突变酶中未观察到显著变化。将DD1的酪氨酸-55定点突变为苯丙氨酸产生了一种无活性的蛋白质，表明该残基具有重要的催化作用。因此，催化酪氨酸残基之前位置的一个残基可能在确定底物和抑制剂的方向中起关键作用。
