Xue L A, Talalay P, Mildvan A S
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Biochemistry. 1990 Aug 14;29(32):7491-500. doi: 10.1021/bi00484a019.
delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position using Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149]. On deuteration of the 4 beta-position (97.0%) of the substrate, kcat(H)/kcat(4 beta-D) is 6.1 in H2O and 6.3 in D2O. The solvent isotope effect, kcat(H2O)/kcat(D2O), is 1.6 for both the 4 beta-H and 4 beta-D substrates. Mutation of Tyr-55 to Phe lowers kcat 4.3-fold; kcat(H)/kcat/4 beta-D) is 5.3 in H2O and 5.9 in D2O, and kcat(H2O)/kcat(D2O) with the 4 beta-H and 4 beta-D substrates is 1.5 and 1.7, respectively, indicating concerted general acid-base catalysis in either the enolization or the ketonization step of both the wild-type and the Tyr-55----Phe (Y55F) mutant enzymes. An additional slow step occurs with the Y55F mutant. Smaller isotope effects on Km are used to estimate individual rate constants in the kinetic schemes of both enzymes. On deuteration of the 4 alpha-position (88.6%) of the substrate, the secondary isotope effect on kcat/Km corrected for composition is 1.11 +/- 0.02 with the wild-type enzyme and 1.12 +/- 0.02 with the Y55F mutant. These effects decrease to 1.06 +/- 0.01 and 1.07 +/- 0.01, respectively, when the 4 beta-position is also deuterated, thereby establishing these to be kinetic (rather than equilibrium) secondary isotope effects and to involve a proton-tunneling contribution. Deuteration of the 6-position of the substrate (92.0%) produces no kinetic isotope effects on kcat/Km with either the wild-type (1.00 +/- 0.01) or the Y55F mutant (1.01 +/- 0.01) enzyme. Since a change in hybridization from sp3 to sp2 occurs at C-4 only during enolization of the substrate and a change in hybridization at C-6 from sp2 to sp3 occurs only during reketonization of the dienol intermediate, enolization of the substrate constitutes the concerted rate-limiting step. Concerted enolization is consistent with the right angle or antarafacial orientations of Tyr-14 and Asp-38 with respect to the enzyme-bound substrate and with the additive effects on kcat of mutation of these catalytic residues [Kuliopulos, A., Talalay, P., & Mildvan, A. S. (1990) Biophys. J. 57, 39a].
δ5-3-酮类固醇异构酶（EC 5.3.3.1）通过将4β-质子以保守的互变异构转移至6β-位，利用Tyr-14作为广义酸、Asp-38作为广义碱，催化δ5-3-酮类固醇异构化为δ4-3-酮类固醇[库利奥普洛斯，A.，米尔德万，A. S.，肖特尔，D.，& 塔拉莱，P.（1989年）《生物化学》28卷，第149页]。在底物的4β-位氘化（97.0%）后，kcat(H)/kcat(4β-D)在H2O中为6.1，在D2O中为6.3。溶剂同位素效应，kcat(H2O)/kcat(D2O)，对于4β-H和4β-D底物均为1.6。将Tyr-55突变为Phe使kcat降低4.3倍；kcat(H)/kcat(4β-D)在H2O中为5.3，在D2O中为5.9，并且4β-H和4β-D底物的kcat(H2O)/kcat(D2O)分别为1.5和1.7，表明野生型和Tyr-55→Phe（Y55F）突变酶在烯醇化或酮化步骤中均存在协同的广义酸碱催化。Y55F突变体还存在一个额外的慢步骤。利用对Km较小的同位素效应来估计两种酶动力学方案中的各个速率常数。在底物的4α-位氘化（88.6%）后，校正组成后的对kcat/Km的二级同位素效应，野生型酶为1.11±0.02，Y55F突变体为1.12±0.02。当4β-位也氘化时，这些效应分别降至1.06±0.01和1.07±0.01，从而确定这些为动力学（而非平衡）二级同位素效应且涉及质子隧穿贡献。底物6-位的氘化（92.0%）对野生型（1.00±0.01）或Y55F突变体（1.01±0.01）酶的kcat/Km均无动力学同位素效应。由于仅在底物烯醇化过程中C-4处的杂化从sp3变为sp2，且仅在二烯醇中间体再酮化过程中C-6处的杂化从sp2变为sp3，底物的烯醇化构成协同的限速步骤。协同烯醇化与Tyr-14和Asp-38相对于酶结合底物的直角或异面取向以及这些催化残基突变对kcat的加和效应一致[库利奥普洛斯，A.，塔拉莱，P.，& 米尔德万，A. S.（1990年）《生物物理杂志》57卷，第39a页]。
