MECHANISTIC PROBES OF PHOSPHORIBOSYLTRANSFERASES

描述：申请者已合成并表征 5-磷酸核糖的非水解性C-糖苷类似物 1-α-二磷酸(PRPP)，并确定它是有竞争力的 谷氨酰胺、次黄嘌呤-鸟嘌呤(H-G)抑制剂：氨基 磷酸核糖基转移酶(PRTase)，它们不能作为 底物。他们建议使用这些化合物来形成死胡同的三元 PRTase复合体，以前不可能进行的实验，因为没有类似的 可以抵抗异构碳的裂解。为 例如，结合的Mg2+和Mn2+离子的数量由 形成全息络合物，其化学计量比将由中子确定 激活分析。酵母菌动力学机制的恢复过渡 随着[Mg~(2+)]的升高，从有序序列到乒乓球的旋转将 也可以通过使用31P核磁共振的反转转移实验和通过 ~(32)PPI-~(32)P-PRPP的平衡同位素交换； 将描述PRPP和二磷酸之间的动力学 可能与5-磷酸核糖结合的酪氨酸酪氨酸酶的“半反应” 氧化碳化中间体。他们为此提出了一个机械的方案。 酶，由此动力学机制的表面转换仅导致 从PPI的发布和绑定之间的速度竞争 奥罗特。 他们建议对假定的氧化碳化作用的可能作用进行评估 中间体沿旋转蛋白酪氨酸酶(和其他)的反应坐标 测试酶上可能的中间体的各种类似物。这些 化合物是5-O-磷酰亚胺核糖的衍生物，它包含 “反应中心”的正电荷和pi性质。作为一个完整的 作为对这些研究的补充，他们还提出了新的双取代类似物 被评估以测试SN2型三元络合物存在于或 在过渡国家附近。嘌呤核苷也是一个类似的例子 磷酸化酶也将被研究。

DESCRIPTION: The applicants have synthesized and characterized non-hydrolyzable C-glycoside analogs of 5-phosphorylribose 1-alpha-diphosphate (PRPP) and determined that it is a competent competitive inhibitor of orotate, hypoxanthine-guanine (H-G) and glutamine:amido phosphoribosyltransferases (PRTases) and that they cannot serve as substrates. They propose to use these compounds to form dead-end ternary complexes of PRTases, previously impossible experiments since no analogs were available that could resist cleavage at the anomeric carbon. For example, the number of bound Mg2+ and Mn2+ ions will be determined by forming the holo complex whose stoichiometry will be determined by neutron activation analysis. The resumed transition in kinetic mechanism of yeast orotate PRTase from ordered sequential to ping pong as [Mg2+] is raised will also be investigated by inversion transfer experiments using 31P NMR and by equilibrium isotope exchange of 32PPi---32P-PRPP; these proposed studies would characterize the dynamics between PRPP and diphosphate in the first "half-reaction" of orotate PRTase that might bind a 5-phosphorylribosyl oxocarbocation intermediate. They propose a mechanistic scheme for this enzyme whereby the apparent transition in kinetic mechanism results only from a competition in rate between the release of PPi and binding of orotate. They propose an evaluation of the likely role of a putative oxocarbocation intermediate along the reaction coordinate of orotate PRTase (and others) by testing various analogs of the likely intermediate on the enzyme. These compounds are derivatives of 5-O-phosphoiminoribose, which contain both positive charge and pi character at the "reactive center". As a full complement to these studies they also propose that new bisubstrate analogs be evaluated to test the idea that an Sn2-type ternary complex exists at or near the transition-state. A similar example for purine nucleoside phosphorylase will also be investigated.