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还不清楚。在这里，我们通过并行分析酶活性部位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高温微生物的(β/α)8桶蛋白质吲哚甘油磷酸合成酶(IGPS)和咪唑甘油磷酸合成酶(ImGPS)的环化酶亚单位(HISF)。对于嗜热酶和中温酶，将通过在不同温度下的预稳态动力学测量来确定各个催化步骤的速率、底物和产物的结合和释放以及化学转化，以获得详细的酶机理图。从这些实验中得出的信息将与环路运动的动力学相关联。为此，中温和高温IGPS活性中心的β1α1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。对于中温和高温HISF，活性中心附近的β/α环的运动将通过核磁共振弛豫色散实验来分析。我们的结果应该允许我们对蛋白质动力学和酶催化中跨越能量障碍之间的耦合的分子本质有新的见解。它们还应该揭示同源的中温和高温(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对酶工程也是有价值的。

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.