Coordination of Protein Dynamics and Chemistry in PNP

PNP 中蛋白质动力学和化学的协调

• 批准号: 7616247
• 负责人: Vern L. Schramm
• 金额: $ 21.09万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7616247
• 关键词: Affinity; Amino Acids; Binding; Biochemical Reaction; Catalysis; Catalytic Domain; Chemicals; Chemistry; Clinical Trials; Collaborations; Complex; Complex Mixtures; Condition; Core Facility; Electronics; Electrons; Enzymes; Equilibrium; Equipment; Fluorescence; Goals; Human; Hydroxyl Radical; Immucillin-H; Ions; Isotopes; Kinetics; Label; Lasers; Left; Ligands; Link; Malignant Neoplasms; Methods; Molecular Conformation; Monitor; Motion; Mutation; Nature; Nucleosides; Oxygen; Personal Satisfaction; Positioning Attribute; Potential Energy; Property; Protein Conformation; Protein Dynamics; Proteins; Protons; Purine-Nucleoside Phosphorylase; Rate; Reaction; Reporting; Research; Role; Signal Transduction; Signaling Protein; Site; Son; Structure; System; T-Cell Leukemia; Temperature; Testing; Time; Tryptophan; analog; base; clinically relevant; computational chemistry; design; enthalpy; infrared spectroscopy; inorganic phosphate; insight; interest; mutant; novel; oxypurine; programs; protein structure; temperature jump; uptake; vibration

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.

嘌呤核苷磷酸化酶(PNP)催化6-氧嘌呤核苷和脱氧核苷的磷酸化。由于动力学同位素效应，过渡态结构是类氧碳正离子，由此设计的过渡态类似物(免疫粘菌素)与PM亲和力结合。晶体结构已经用底物、产物和过渡态类似物进行了解析。新出现的催化假说是草卡宾离子的形成。 从核糖基的5‘-羟基与酶结合的磷酸盐亲核试剂相互作用形成的相邻基团的过渡态。催化中心放置邻位氧基核糖04‘，辅助电子贡献从核糖基到离开基团。这种几何构型支持“电子促进振动”，其中蛋白质基团波动以拉近氧原子，从而促进电子排出。计算化学动力学(施瓦茨，项目4)将确定与这种动力学相关的基团。预测催化位点突变将扰乱 并进行了激振试验。同位素编辑红外光谱学(Callender，Project 1)已经建立了与磷酸盐亲核分子和离开基团相互作用相关的强光谱带。我们提出了时间分辨光谱分析来关联蛋白质动力学、催化位置化学、pH、离开基团和亲核分子相互作用的变化。激光将在快速时间尺度上诱导动态平衡混合物PNP与底物和产物的T跳跃，然后对每个参数进行时间分辨监测。笼状H+将被用来引发pH跃迁，以检查通过质子供体/受体位置的化学和结构扰动。笼状磷酸盐将用于将PNP、免疫粘菌素转化为PNP、免疫粘菌素.PO4，然后在相关的结构变化之后进行同位素编辑 具有缓慢起效的紧密结合，类似于过渡态复合体(Dyer，Project 3)。时间分辨光谱将从PSEC到MIN时间尺度进行检查，以跟踪局部和全球动态。初步结果为PO4和离开基团相互作用建立了丰富的红外光谱特征。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。