STRUCTURE/FUNCTION RELATIONSHIPS OF HPPK

这项多学科研究的目的是阐明6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK)的结构与功能关系，它是叶酸生物合成途径中的第一个酶。我们的系统是重组大肠杆菌HPPK，因为大肠杆菌同工酶是研究酶促焦磷酰化转移机理的最佳模型酶。这些结果将提供关于HPPK的结构以及它如何催化焦磷酸转移的详细信息。这项拟议的研究对酶学具有重要意义，因为人们对焦磷酸酶的结构和机制知之甚少。此外，从拟议的研究中获得的结构和机制信息将对HPPK抑制剂的设计至关重要。由于大多数微生物必须从头合成叶酸，而人和动物不能也不需要合成叶酸，HPPK抑制剂可能成为新的抗菌剂。这具有重大的生物医学意义，特别是考虑到近年来抗生素耐药性的迅速增加，使得目前的抗生素对许多微生物感染无效，并造成了全球卫生保健危机。针对L，将通过稳态动力学、瞬时动力学、热力学和立体化学分析来确定HPPK催化反应的动力学途径和热力学。在特定目标2中，定点突变，结合突变体的生化和生物物理特性，将用于鉴定活性位点残基，并阐明它们在底物结合和催化中的作用。在具体目标3中，将使用生物物理方法评估突变对HPPK在自由态和底物结合态的结构和稳定性的影响。在特定目标4中，将通过双共振和三重共振多维核磁共振来确定游离HPPK的溶液结构。在具体目标5中，将使用核磁共振来鉴定活性中心残基，并确定HPPK与底物类似物的络合物的结构。

The goal of the proposed multidisciplinary research is to elucidate the structure-function relationships of 6-hydroxymethyl-7,8- dihydropterin pyrophosphokinase (HPPK), the first enzyme in the folate biosynthesis pathway. Our system is recombinant E. coli HPPK because the E. coli isozyme is the best model enzyme for studying the mechanisms of enzymatic pyrophosphoryl transfer. The results will provide the detailed information on the structure of HPPK and how it catalyzes the transfer of pyrophosphate. The proposed research is of fundamental importance to enzymology because very little is known about the structures and mechanisms of any pyrophosphokinases. Furthermore, the structural and mechanistic information that will be obtained from the proposed research will be essential for design of inhibitors for HPPK. Since most microorganisms must synthesize folate de novo but man and animals cannot and don't need to, HPPK inhibitors may become new antimicrobial agents. This is of great biomedical significance especially in light of rapidly increasing antibiotic resistance in recent years that has rendered the current antibiotics ineffective for treating many microbial infections and caused a worldwide health care crisis. In Specific Aim l, the kinetic pathway and energetics of the HPPK-catalyzed reaction will be determined by steady-state kinetic, transient kinetic, thermodynamic and stereochemical analyses. In Specific Aim 2, site-directed mutagenesis, in conjunction with biochemical and biophysical characterizations of the mutants, will be used to identify the active-site residues and to elucidate their roles in substrate binding and catalysis. In Specific Aim 3, biophysical methods will be used to assess the effects of mutations on the structure and stability of HPPK at both free and substrate-bound states. In Specific Aim 4, the solution structure of the free HPPK will be determined by double-and triple-resonance multidimensional NMR. In specific Aim 5, NMR will be used to identify the active-site residues and to determine the structures of HPPK in complex with substrate analogues.