Biochemistry and Molecular Biology Program, Department of Chemistry, University of Richmond, Richmond, Virginia 23173, USA.
Protein Sci. 2010 Oct;19(10):1820-9. doi: 10.1002/pro.459.
Bovine glutamate dehydrogenase (GDH) is allosterically regulated and requires substrate-induced subunit interactions for maximum catalytic activity. Steady-state and presteady-state kinetics indicate that the rate-limiting step depends on the nature of the substrate and are likely associated with conformational fluctuations necessary for optimal hydride transfer. Deuterated glutamate shows a steady-state isotope effect but no effect on the presteady-state burst rate, demonstrating that conformational effects are rate limiting for hydride transfer while product release is overall rate limiting for glutamate. Guanidine hydrochloride unfolding, heat inactivation, and differential scanning calorimetry demonstrate the effects of alternative substrates, glutamate and norvaline, on conformational stability. Glutamate has little effect on overall stability, whereas norvaline markedly stabilizes the protein. Limited proteolysis demonstrates that glutamate had a variety of effects on local flexibility, whereas norvaline significantly decreased conformational fluctuations that allow protease cleavage. Dynamic light scattering suggests that norvaline stabilizes all interfaces in the hexamer, whereas glutamate had little effect on trimer-trimer interactions. The substrate glutamate exhibits negative cooperativity and complex allosteric regulation but has only minor effects on global GDH stability, while promoting certain local conformational fluctuations. In contrast, the substrate norvaline does not show negative cooperativity or allow allosteric regulation. Instead, norvaline significantly stabilizes the enzyme and markedly slows or prevents local conformational fluctuations that are likely to be important for cooperative effects and to determine the overall rate of hydride transfer. This suggests that homotropic allosteric regulation by the enzymatic substrate involves changes in both global stability and local flexibility of the protein.
牛谷氨酸脱氢酶（GDH）是变构调节的，需要底物诱导的亚基相互作用以达到最大的催化活性。稳态和预稳态动力学表明，限速步骤取决于底物的性质，并且可能与最佳氢转移所需的构象波动有关。氘代谷氨酸显示出稳态同位素效应，但对预稳态爆发速率没有影响，这表明构象效应是氢转移的限速步骤，而产物释放是谷氨酸的整体限速步骤。盐酸胍展开、热失活和差示扫描量热法证明了替代底物谷氨酸和正缬氨酸对构象稳定性的影响。谷氨酸对整体稳定性几乎没有影响，而正缬氨酸则显著稳定蛋白质。有限蛋白酶解表明，谷氨酸对局部灵活性有多种影响，而正缬氨酸则显著降低了允许蛋白酶切割的构象波动。动态光散射表明，正缬氨酸稳定六聚体中的所有界面，而谷氨酸对三聚体-三聚体相互作用几乎没有影响。底物谷氨酸表现出负协同性和复杂的变构调节，但对全局 GDH 稳定性只有很小的影响，同时促进某些局部构象波动。相比之下，底物正缬氨酸不表现出负协同性或允许变构调节。相反，正缬氨酸显著稳定酶并显著减缓或阻止可能对协同效应很重要并决定氢转移整体速率的局部构象波动。这表明酶底物的同变变构调节涉及蛋白质整体稳定性和局部灵活性的变化。
