Coordination of Protein Dynamics and Chemistry in PNP

嘌呤核苷磷酸化酶（PNP）催化6-氧代嘌呤核苷和脱氧核苷的磷酸解。过渡态结构是oxacarbenium样的动力学同位素效应和过渡态类似物（Immucillins）设计从这个结构结合与pM亲和力。晶体结构已解决与底物，产品和过渡态类似物。关于催化作用的假说是氧碳烯鎓离子的形成 通过来自核糖基的5 '-羟基和酶结合的磷酸亲核试剂的相邻基团相互作用的过渡态。催化位点将邻近的氧置于核糖基04 '，帮助电子从核糖基贡献到离去基团。这种几何结构支持“电子促进振动”，其中蛋白质基团波动以使氧更接近，促进电子排出。计算化学动力学（Schwartz，项目4）将确定与此动态相关的基团。预测催化位点突变会破坏 将制作并测试促进振动。同位素编辑红外光谱（Callender，项目1）已经建立了与磷酸盐亲核体和离去基团相互作用相关的强光谱带。我们提出时间分辨光谱分析，以关联蛋白质动力学，催化位点化学，pH值，离去基团和亲核相互作用的变化。用激光在快速时间尺度上诱导PNP与底物和产物的动态平衡混合物的T跃变，然后对每个参数进行时间分辨监测。笼状H+将用于启动pH跳跃，以检查通过质子供体/受体位点的化学和结构扰动。笼状磷酸盐将用于将PNP.Immucillin转化为PNP.Immucillin.PO4，然后在相关结构变化后进行同位素编辑 具有缓慢开始的紧密结合以类似于过渡态复合物（Dyer，项目3）。时间分辨光谱将检查从皮秒到分钟的时间尺度，以遵循本地和全球的动态。初步结果建立了丰富的红外光谱特征PO 4和离去基团的相互作用。这些结果将为蛋白质动力学、配体相互作用和催化动力学之间的关系提供新的见解。

Purine nucleoside phosphorylase (PNP) catalyzes phosphorolysis of 6-oxypurine nucleosides and deoxynucleosides. The transition state structure is oxacarbenium-like from kinetic isotope effects and transition state analogues (Immucillins) designed from this structure bind with pM affinity. Crystal structures have been solved with substrate, product and transition state analogues. The hypothesis emerging for catalysis is formation of an oxacarbenium ion transition state by neighboring group interactions from the 5'-hydroxyl of the ribosyl group and the enzyme-bound phosphate nucleophile. The catalytic site places neighbor oxygens the ribosyl 04', assisting electron contribution from the ribosyl group to the leaving group. This geometry supports an 'electronic promoting vibration' where protein groups fluctuate to bring oxygens closer, promoting electron expulsion. Computational chemistry dynamics (Schwartz, Project 4) will identify groups associated with this dynamic. Catalytic site mutations predicted to disrupt the promoting vibration will be made and tested. Isotope-edited infrared spectroscopy (Callender, Project 1) has established strong spectral bands associated with the phosphate nueleophile and the leaving group interactions. We propose time-resolved spectral analysis to correlate changes in protein dynamics, catalytic site chemistry, pH, leaving group and nucleophile interactions. T-jumps of dynamic equilibrium mixtures PNP with substrates and products will be induced by laser on a fast time scale followed by time-resolved monitoring of each parameter. Caged H+ will be used to initiate pH jumps to examine chemical and structural perturbations through proton donor/acceptor sites. Caged phosphate will be used to convert PNP.Immucillin to PNP.Immucillin.PO4, followed by isotope-edited following of the structural changes associated with slow-onset tight binding to resemble a transition state complex (Dyer, Project 3). Time-resolved spectra will be examined from psec to min time scales to follow local and global dynamics. Preliminary results establish rich IR spectral signatures for PO4 and leaving group interactions. These results will provide novel insights for the relationship between protein dynamics, ligand interactions and dynamics in catalysis.