Altered Enthalpy-Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase
Altered Enthalpy-Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase
复制标题
DOI：
10.1021/bi9005896
复制
发表时间：
2009-06-16
期刊：
影响因子：
2.9
通讯作者：
Schramm, Vern L.
中科院分区：
文献类型：
--
作者：
Edwards, Achelle A.;Mason, Jennifer M.;Schramm, Vern L.
人嘌呤核苷磷酸化酶（PNP）属于三聚体类PNP，在脱氧鸟苷的分解代谢中起重要作用。人类PNP的遗传缺陷导致特异性t细胞免疫缺陷，PNP的过渡态类似抑制剂正在开发中，用于治疗t细胞癌和自身免疫性疾病。已经开发了四代Immucillins，每一代都含有与人PNP具有小摩尔亲和力的抑制剂。PNP的完全抑制发生在与三个亚基中的第一个结合时，与后续位点的结合发生负协同性。相比之下，底物模拟物和产物结合没有协同性。用等温量热法滴定人体PNP表明，结构刚性的第一代Immucillin （K-d = 56 pM）的结合是由大的负焓值（δ H = -21.2 kcal/mol）和大量的熵（-T δ S）惩罚驱动的。结合最紧密的抑制剂（K-d = 5- 9pm）具有增加的构象灵活性。尽管它们在溶液中的构象自由，柔性抑制剂结合具有高亲和力，因为减少了熵惩罚。熵惩罚是由pnp -抑制剂复合物的构象冻结引起的，熵项由蛋白质动力学主导。构象柔性免疫蛋白减少了系统的熵罚。破坏过渡态类似物与PNP的核糖基5′-羟基相互作用会产生有利的结合熵。17种免疫蛋白紧密结合的特点是焓贡献大，强调了它们与过渡态的相似性。通过在抑制剂结构中引入灵活性，改变了焓熵补偿模式，以允许更紧密的结合。
Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (K-d = 56 pM) is driven by large negative enthalpy values (Delta H = -21.2 kcal/mol) with a substantial entropic (-T Delta S) penalty. The tightest-binding inhibitors (K-d = 5-9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP-inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5'-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the 17 Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. Via introduction of flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.