Kuliopulos A, Mullen G P, Xue L, Mildvan A S
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Biochemistry. 1991 Apr 2;30(13):3169-78. doi: 10.1021/bi00227a003.
The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)
已证明由δ5-3-酮甾体异构酶催化的反应是通过δ5-3-酮甾体底物的协同烯醇化作用形成双烯醇中间体来进行的，这一过程由Tyr-14引发，它与3-酮基形成氢键并使其质子化，同时Asp-38在相同的限速步骤中从底物的C-4位去除一个轴向（β）质子[薛，L.，塔拉莱，P.，& 米尔德万，A.S.（1990）《生物化学》29，7491 - 7500；库利奥普洛斯，A.，米尔德万，A.S.，肖特尔，D.，& 塔拉莱，P.（1989）《生物化学》26，3927 - 3937]。由于轴向C-4质子是由Asp-38从底物上方去除的，因此要确定这种快速协同烯醇化的完整立体化学，需要了解Tyr-14接近酶结合甾体的方向信息。制备了双突变酶Y55F + Y88F，它保留Tyr-14作为唯一的Tyr残基，与野生型酶相比，对于δ5-雄烯-3,17-二酮，其kcat仅降低了4.5倍（12,000 s-1），KM降低了3.6倍（94 microM）。对10个Phe残基的芳香环进行氘代进一步有助于在600 MHz的TOCSY谱中归属Tyr-14的芳香质子共振峰，其化学位移为6.66 ± 0.01 ppm（3,5H）和6.82 ± 0.01 ppm（2,6H）。pH从4.9变化到10.9并未改变这些化学位移，表明Tyr-14的pKa超过10.9。归属给三个His残基的共振峰的滴定pKa值与野生型酶的非常相似。19-去甲睾酮（一种反向异构酶反应的产物类似物和底物）的结合分别使Tyr-14的3,5-和2,6-质子共振峰发生了-0.12和-0.06 ppm的向低场位移，这可能是由于甾体的3-酮基去屏蔽作用导致的，同时还使10个Phe残基中的8个的化学位移发生了+0.29至-0.41 ppm的变化，以及12个环位移甲基共振峰中的10个发生了较小变化，表明甾体诱导了酶的构象变化。H2O中的NOESY谱显示，Tyr-14的3,5-质子共振峰与结合甾体的重叠的2α-、2β-或6β-质子共振峰之间有很强的负Overhauser效应，但与甾体的4-或6α-质子之间没有NOE效应。（摘要截断于400字）
