Zhou X, Kay S, Toney M D
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 1998 Apr 21;37(16):5761-9. doi: 10.1021/bi973010u.
The pyridoxal phosphate (PLP) dependent enzyme dialkylglycine decarboxylase (DGD) specifically binds alkali metal ions near the active site. Large ions (Rb+, K+) activate the enzyme while smaller ones (Na+, Li+) inhibit it. Crystallographic results have shown that DGD undergoes a metal ion size dependent structural switch [Hohenester, E., Keller, J. W., and Jansonius, J. N. (1994) Biochemistry 33, 13561], but no evidence for multiple conformations in crystalline DGD was obtained. Here, evidence is presented that DGD-K+ in solution exists in two conformations differing in catalytic competence. Initial rate traces for DGD-K+ exhibit a high degree of curvature due to decreasing activity over time. DGD remains tetrameric under the assay conditions as demonstrated by gel filtration experiments, arguing against the possibility of subunit dissociation as the source of activity loss. Likewise, the mass spectrum of DGD shows a single covalent form. A hysteretic model that assumes two slowly interconverting enzyme forms accounts well for the initial rate data when kinetic parameters from biphasic pre-steady-state kinetics are employed. The fit of the model to the data yields an estimate of 59 +/- 1% for the fast form. A cooperative model cannot account for the data. Double reciprocal plots for coenzyme binding to DGD exhibit two linear phases. Similarly, two kinetic phases are observed in PLP association kinetics. The substitution of Na+ or Rb+ for K+ alters the steady-state kinetic parameters of DGD. Preincubation of DGD-K+ with the competitive inhibitor 1-aminocyclopropane-1-carboxylate (ACC) lowers both kcat and KAIB apparently by drawing the enzyme toward the less active, tighter binding form observed in the pre-steady-state kinetics. These results suggest that the structure of the protein around the alkali metal ion determines the conformational distribution. The transamination reaction with l-alanine was coupled in the pre-steady-state to the LDH-catalyzed oxidation of NADH. This experiment yields an estimate of 68 +/- 4% for the fast form, in agreement with the hysteretic fit to the steady-state data. The reaction of DGD with dithiobis(nitrobenzoate) was used to probe the preexisting forms of DGD. Preincubation of DGD with ACC, like the exchange of Na+ for K+, shifts the conformational distribution, in agreement with the steady-state kinetics. These experiments clearly demonstrate that DGD is a hysteretic enzyme whose conformational distribution is controlled by the identity of the alkali metal ion bound near the active site, and that cooperativity does not play a role in catalysis or regulation.
磷酸吡哆醛（PLP）依赖性酶二烷基甘氨酸脱羧酶（DGD）在活性位点附近特异性结合碱金属离子。大离子（Rb⁺、K⁺）激活该酶，而较小的离子（Na⁺、Li⁺）则抑制它。晶体学结果表明，DGD会发生金属离子大小依赖性的结构转换[霍内斯特，E.，凯勒，J. W.，和扬松纽斯，J. N.（1994年）《生物化学》33卷，13561页]，但未获得结晶DGD中存在多种构象的证据。在此，有证据表明溶液中的DGD-K⁺以两种催化活性不同的构象存在。由于活性随时间降低，DGD-K⁺的初始速率曲线呈现出高度的曲率。凝胶过滤实验表明，在测定条件下DGD保持四聚体状态，这排除了亚基解离作为活性丧失来源的可能性。同样，DGD的质谱显示为单一的共价形式。当采用双相预稳态动力学的动力学参数时，一个假设两种缓慢相互转化的酶形式的滞后模型能很好地解释初始速率数据。该模型与数据的拟合得出快速形式占比为59±1%。协同模型无法解释这些数据。辅酶与DGD结合的双倒数图呈现出两个线性阶段。类似地，在PLP结合动力学中也观察到两个动力学阶段。用Na⁺或Rb⁺替代K⁺会改变DGD稳态动力学参数。用竞争性抑制剂氨基环丙烷-1-羧酸（ACC）对DGD-K⁺进行预孵育，显然会使kcat和KAIB降低，这可能是因为将酶推向了在预稳态动力学中观察到的活性较低、结合更紧密的形式。这些结果表明，碱金属离子周围蛋白质的结构决定了构象分布。在预稳态下，将与L-丙氨酸的转氨反应与乳酸脱氢酶催化的NADH氧化反应偶联。该实验得出快速形式占比为68±4%，与稳态数据的滞后拟合结果一致。利用DGD与二硫代双（硝基苯甲酸）的反应来探究DGD的预先存在形式。与用Na⁺替代K⁺一样，用ACC对DGD进行预孵育会改变构象分布，这与稳态动力学结果一致。这些实验清楚地表明，DGD是一种滞后酶，其构象分布受活性位点附近结合的碱金属离子种类控制，并且协同作用在催化或调节过程中不起作用。
