Golicnik Marko, Olguin Luis F, Feng Guoqiang, Baxter Nicola J, Waltho Jonathan P, Williams Nicholas H, Hollfelder Florian
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
J Am Chem Soc. 2009 Feb 4;131(4):1575-88. doi: 10.1021/ja806421f.
The isomerization of beta-glucose-1-phosphate (betaG1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (betaPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (betaG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (kcat = 65 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.7 microM, Ki = 122 microM). If alphaG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (kcat = 74 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.8 microM, Ki = 122 microM, K(alphaG16BP) = 91 microM for productive binding, K(alphaG16BP) = 21 microM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140,000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the betaPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a betaG1P x MgF3(-) x betaPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M(5) and suggests an affinity of the MgF3(-) moiety to this transition-state analogue (TSA) of < or = 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of betaPGM.
已使用稳态和预稳态动力学分析研究了由β - 磷酸葡萄糖变位酶（βPGM）催化的β - 葡萄糖 - 1 - 磷酸（βG1P）异构化为β - 葡萄糖 - 6 - 磷酸（G6P）的过程。在低浓度的β - 葡萄糖 - 1,6 - 二磷酸（βG16BP）存在下，反应通过乒乓双底物机制进行，存在底物抑制（kcat = 65 s(-1)，K(βG1P) = 15 μM，K(βG16BP) = 0.7 μM，Ki = 122 μM）。如果使用αG16BP作为辅因子，则会观察到更复杂的动力学行为，但非线性进程曲线可以拟合以揭示更多催化参数（kcat = 74 s(-1)，K(βG1P) = 15 μM，K(βG16BP) = 0.8 μM，Ki = 122 μM，对于有效结合K(αG16BP) = 91 μM，对于无效结合K(αG16BP) = 21 μM）。这些数据表明，底物结构的变化对过渡态亲和力（以速率加速计约为140,000倍）的影响远大于基态结合（以结合亲和力计为110倍）。当存在氟化物和镁离子时，会观察到βPGM的时间依赖性抑制。从拟合这些进程曲线获得的参数的浓度依赖性表明，在反应条件下形成了βG1P x MgF3(-) x βPGM抑制复合物。该复合物的整体稳定性常数约为2 x 10(-16) M(5)，表明MgF3(-)部分对这种过渡态类似物（TSA）的亲和力≤70 nM。详细的动力学分析表明了一种在溶液中不存在的特殊类型的TSA是如何在酶的活性位点组装的。进一步的实验表明，在先前结构研究的条件下，磷酸化葡萄糖仅在作为TSA与酶结合时才会持续存在。存在氟化物时对TSA形成的偏好以及不存在氟化物时底物的水解，排除了在βPGM活性位点形成稳定的五价磷烷中间体的可能性。
