呈碎片状的底物：亚磷酸二阴离子对3-磷酸甘油脱氢酶（NAD⁺）的变构激活作用
A substrate in pieces: allosteric activation of glycerol 3-phosphate dehydrogenase (NAD+) by phosphite dianion.
作者信息
Tsang Wing-Yin, Amyes Tina L, Richard John P
机构信息
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA.
出版信息
Biochemistry. 2008 Apr 22;47(16):4575-82. doi: 10.1021/bi8001743. Epub 2008 Apr 1.
The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD (+), GPDH) saturated with NADH is (1.0 x 10 (6) M (-1) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 1.1 x 10 (8), which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH ( K d > 0.1 M), but the bound dianion strongly activates GLY toward enzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M (-2) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 5 x 10 (5) M (-1), which was used to calculate an intrinsic phosphite binding energy of -7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structural data provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring (1) a binding pocket complementary to the reactive substrate fragment which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.
用甘油3 - 磷酸脱氢酶（NAD(+)，GPDH）饱和NADH还原磷酸二羟丙酮（DHAP）和中性截短底物乙醇醛（GLY）的二级速率常数之比为(1.0×10⁶ M⁻¹ s⁻¹)/(8.7×10⁻³ M⁻¹ s⁻¹) = 1.1×10⁸，该比值用于计算至少为11.0 kcal/mol的内在磷酸结合能。亚磷酸二阴离子与GPDH的结合非常弱（Kd > 0.1 M），但结合的亚磷酸二阴离子能强烈激活GLY，使其被NADH进行酶促还原。因此，大的内在亚磷酸结合能仅在GPDH催化反应的过渡态时才表现出来。亚磷酸激活的和未激活的GPDH催化NADH还原GLY的速率常数之比为(4300 M⁻² s⁻¹)/(8.7×10⁻³ M⁻¹ s⁻¹) = 5×10⁵ M⁻¹，该比值用于计算亚磷酸二阴离子与GPDH催化还原GLY的过渡态复合物缔合时的内在亚磷酸结合能为 -7.7 kcal/mol。现已表明，亚磷酸二阴离子能激活结合的底物进行酶促质子转移、脱羧、氢化物转移和磷酰基转移反应。结构数据提供了有力证据，表明亚磷酸二阴离子结合引起的酶促激活发生在一个模块化活性位点，该位点具有：(1) 一个与反应性底物片段互补的结合口袋，其中包含催化底物片段或整个底物反应所需的所有活性位点残基；(2) 一个磷酸/亚磷酸二阴离子结合口袋，通过柔性蛋白质环的移动来包围非反应性氧阴离子从而形成该口袋。我们提出，亚磷酸二阴离子和/或磷酸二阴离子底物结合时伴随的环运动和相关蛋白质构象变化导致底物和/或其片段被包裹在蛋白质内部，并使活性位点残基处于能为催化反应的过渡态提供最佳稳定作用的位置。
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