Linkage between catalytic mechanism and conformational dynamics in (beta/alpha)8-barrel enzymes

酶的催化能力与其构象动力学有关，但这种联系的分子决定因素在很大程度上是不清楚的。在这里，我们通过并行分析酶活性位点附近的动力学机制和环的动力学来解决这个问题。我们将使用来自中温和嗜热微生物的（β / α）8桶甘油磷酸合成酶（IGPS）蛋白和咪唑甘油磷酸合成酶（ImGPS）环化酶亚基（HisF）。对于嗜热和中温酶，单个催化步骤的速率，底物和产物的结合和释放，化学转化，将通过在不同温度下使用稳态前动力学测量来确定，以获得酶机制的详细图像。然后，从这些实验中推断出的信息将与环运动的动力学相关联。为此，亲中温和嗜热IGPS活性位点的beta1alpha1环将被自旋标记，其在催化过程中的动力学变化将被EPR跟踪。对于亲中温和嗜热的HisF，将通过核磁共振弛豫色散实验分析活性位点附近的β / α环的运动。我们的结果应该使我们对酶催化中蛋白质动力学和跨越能量屏障之间耦合的分子性质有了新的认识。他们还应该揭示同源的亲中温和亲热（β / α）8桶酶在催化策略上是否以及如何不同。更好地理解动力学和催化效率之间的关系也将对酶工程有价值。

The catalytic power of enzymes is linked with its conformational dynamics, but the molecular determinants of this linkage are largely unclear. Here, we address this problem by analyzing in parallel the kinetic mechanisms and the dynamics of loops near the active sites of enzymes. We will use the (beta/alpha)8-barrel proteins indoleglycerol phosphate synthase (IGPS) and the cyclase subunit (HisF) of imidazole glycerol phosphate synthase (ImGPS) from mesophilic and thermophilic microorganisms. For the thermophilic and the mesophilic enzymes, the rates of the individual catalytic steps, substrate and product binding and release, chemical transformation, will be determined by using pre-steady state kinetic measurements at various temperatures to obtain a detailed picture of the enzymatic mechanisms. The information deduced from these experiments will then be correlated with the dynamics of loop movements. To this end, the beta1alpha1-loop at the active site of mesophilic and thermophilic IGPS will be spin labeled, and changes in its dynamics in the course of catalysis will be followed by EPR. For mesophilic and thermophilic HisF, motions of the beta/alpha-loops near the active site will be analyzed by NMR relaxation dispersion experiments. Our results should allow us to gain novel insight into the molecular nature of the coupling between protein dynamics and the crossing of energy barriers in enzyme catalysis. They should also reveal whether and how homologous mesophilic and thermophilic (beta/alpha)8-barrel enzymes differ in their catalytic strategies. A better understanding of the relationship between dynamics and catalytic efficiency will also be valuable for enzyme engineering.