Schlegel B P, Jez J M, Penning T M
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Biochemistry. 1998 Mar 10;37(10):3538-48. doi: 10.1021/bi9723055.
Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, E.C. 1.1.1.213, AKR1C9) is a member of the aldo-keto reductase (AKR) superfamily which inactivates circulating steroid hormones. We have proposed a catalytic mechanism in which Tyr 55 acts as a general acid with its pK value being lowered by a hydrogen bond with Lys 84 which is salt-linked to Asp 50. To test this mechanism, residues at the active site were mutated and the mutant enzymes (Y55F, Y55S, K84M, K84R, D50N, D50E, and H117A) were purified to homogeneity from an Escherichia coli expression system. Spectrophotometric assays showed that mutations of Tyr 55 and Lys 84 gave enzymes that were apparently inactive for steroid oxidation and reduction. All mutants appeared inactive for steroid reduction. The catalytic efficiencies for steroid oxidation were reduced 4-10-fold for the Asp 50 mutants and 300-fold for the H117A mutant. Fluorescence titration with NADPH demonstrated that each mutant bound cofactor unimpeded. Equilibrium dialysis indicated that the competitive inhibitor testosterone formed E.NADH.testosterone complexes only with the Y55F, Y55S, and D50N mutants with Kd values 10-fold greater than those for wild-type. Therefore the loss of steroid oxidoreductase activity observed for the Tyr 55 mutants cannot be attributed simply to an inability to bind steroid. Using a highly sensitive radiometric assay in which the conversion of [14C]-5 alpha-dihydrotestosterone (DHT) to [14C]-3 alpha-androstanediol (3 alpha-Diol) was measured, the rate enhancement (kcat/knoncat) for the reaction was estimated to be 2.6 x 10(9). Using this assay, all mutants formed steroid product with decreases in an overall rate enhancement of 10(1)-10(4). It was found that Tyr 55 made the single largest contribution to rate enhancement. This is the first instance where point mutations in the conserved catalytic tetrad of an AKR yielded enzymes which were still catalytically active. This enabled the construction of pH vs kcat profiles for the reduction of [14C]-5 alpha-DHT catalyzed by the tetrad mutants. These profiles revealed that the titratable group assigned to the general acid (pK = 6.50 +/- 0.42) was eliminated in the Y55F and H117A mutants. The pH-independent value of kcat was decreased in the H117A and Y55F mutants, by 2 and 4 log units, respectively. pH vs kcat(app) profiles for the oxidation of [3H]-3 alpha-Diol showed that the same titratable group (pK = 7.50 +/- 0.30) was eliminated in both the Y55F and K84M mutants but was retained in the H117A mutant. Since only the Y55F mutant eliminated the titratable group in both the reduction and oxidation directions it is assigned as the catalytic general acid/base. The differential effects of His 117 and Lys 84 on the ionization of Tyr 55 are explained by a "push-pull" mechanism in which His 117 facilitates proton donation and Lys 84 facilitates proton removal by Tyr 55.
大鼠肝脏3α-羟基类固醇脱氢酶（3α-HSD，E.C. 1.1.1.213，AKR1C9）是醛酮还原酶（AKR）超家族的成员之一，可使循环中的类固醇激素失活。我们提出了一种催化机制，其中Tyr 55作为广义酸，其pK值通过与Lys 84形成氢键而降低，Lys 84与Asp 50形成盐键。为了验证这一机制，对活性位点的残基进行了突变，并从大肠杆菌表达系统中纯化得到了均一的突变酶（Y55F、Y55S、K84M、K84R、D50N、D50E和H117A）。分光光度法分析表明，Tyr 55和Lys 84的突变使酶对类固醇氧化和还原反应明显无活性。所有突变体对类固醇还原反应均无活性。Asp 50突变体的类固醇氧化催化效率降低了4-10倍，H117A突变体降低了300倍。用NADPH进行荧光滴定表明，每个突变体均能不受阻碍地结合辅因子。平衡透析表明，竞争性抑制剂睾酮仅与Y55F、Y55S和D50N突变体形成E.NADH.睾酮复合物，其解离常数Kd值比野生型大10倍。因此，Tyr 55突变体观察到的类固醇氧化还原酶活性丧失不能简单地归因于无法结合类固醇。使用一种高灵敏度的放射性测定法，测量[14C]-5α-二氢睾酮（DHT）向[14C]-3α-雄甾二醇（3α-二醇）的转化，该反应的速率增强（kcat/knoncat）估计为2.6×109。使用该测定法，所有突变体均形成类固醇产物，总体速率增强降低了101-104倍。发现Tyr 55对速率增强的贡献最大。这是首次在AKR保守催化四联体中进行点突变产生仍具有催化活性的酶的实例。这使得能够构建由四联体突变体催化还原[14C]-5α-DHT的pH与kcat关系曲线。这些曲线表明，在Y55F和H117A突变体中，分配给广义酸（pK = 6.50±0.42）的可滴定基团消失。H117A和Y55F突变体中kcat的pH无关值分别降低了2和4个对数单位。[3H]-3α-二醇氧化的pH与kcat（app）关系曲线表明，Y55F和K84M突变体中相同的可滴定基团（pK = 7.50±0.30）消失，但在H117A突变体中保留。由于只有Y55F突变体在还原和氧化方向上都消除了可滴定基团，因此它被指定为催化广义酸/碱。His 117和Lys 8通过一种“推-拉”机制解释了对Tyr 55电离的不同影响，其中His 117促进质子供体作用，Lys 84促进Tyr 55去除质子。
