RABBIT MUSCLE ALDOLASE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。醛缩酶是一种普遍存在于所有生物体内的酶，从原核生物到哺乳动物。它们有分裂碳碳键的能力。它们的作用在糖酵解中最为人所知，其中果糖-1,6-二磷酸（FBP）醛缩酶将FBP裂解为二羟基丙酮磷酸（DHAP）和甘油醛-3-磷酸。由于缺乏真正的反应中间体晶体结构，对残基的催化作用分配受到了阻碍。本课题的目的是研究兔肌醛缩酶的催化机理，该酶是一类果糖-1,6-二磷酸醛缩酶。我们计划用原生和突变醛缩酶晶体进行底物浸泡实验，以深入了解与催化有关的活性位点残基。通过使用不同浸泡时间的组合，我们希望在反应方案中捕获催化反应中间体，已知这涉及3个共价中间体的形成。时间分辨晶体学已成功应用于配体或底物浸泡的兔肌醛缩酶晶体。利用该技术，进一步深入了解醛缩酶的催化机理仍是可能的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aldolase are ubiquitous enzymes found in all organisms, from prokaryotes to mammals. They have the ability to cleave carbon-carbon bonds. Their role is best known in glycolysis where fructose-1,6-bisphosphate (FBP) aldolase cleaves FBP to dihydroxyacetone-phosphate (DHAP) and glyceraldehy-3-phosphate. The catalytic role assigment to residues has beeen hampered by the lack of genuine reaction intermediate crystallographic structures. The aim of this project is to study the catalytic mechanism of the rabbit muscle aldolase, which is a class I fructose-1,6-bisphosphate aldolase. We are planning to use subtrate soaking experiments with native and mutant aldolase crystals to get insight about residues of the active site implicated in the catalysis. By using a combination of diverse soaking time, we want to trap catalytic reaction intermdiates among the reaction scheme, which is known to implicate the formation of 3 covalent intermediates. Time-resolved crystallography applied to rabbit muscle aldolase crystal soaked with ligands or substrates have been successfully used in the past years. Further insight into the catalytic mechanism of aldolase still possible by using this technique.