STRUCTURAL BASIS FOR SUBSTRATE RECOGNITION IN CLASS I TAGATOSE-1,6-BISPHOSPHATE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 I类糖酵解醛缩酶是一种可逆催化双磷酸化己糖裂解的酶，在与赖氨酸残基形成质子化的亚胺后，生成3-磷酸甘油醛(G3P)和二羟丙酮-磷酸(DHAP)。塔格糖-1，6-二磷酸缩醛酶(TBPA)以相似的亲和力裂解塔格糖-1，6-二磷酸(TBP)和果糖-1，6-二磷酸(FBP)，而果糖-1，6-二磷酸缩醛酶(FBPA)则专一于FBP。裂解/缩合产物仅在第三和第四碳原子的立体化学构型方面不同，提出了醛缩合酶如何立体地识别其同源底物的问题。 兔肌肉FBPA在运动学和结构上都有很好的特征。此外，一种亚胺中间体，被困在晶体FBPA的活性部位，最近揭示了这种酶的立体特异性的重要方面。然而，TBPA允许性特异性的结构特征仍不清楚。因此，我们对TBPA的催化中间体进行了X射线结晶学研究，以揭示其活性中心的分子结构以及参与配体识别和催化的残基。为此，我们从化脓性链球菌及其硒甲硫化衍生物中提纯并结晶了天然的TBPA。MAD分期实验已经确定了该酶的三级结构，揭示了一个8桶亚单位折叠。 我们建议高分辨率地确定与TBPA形成的反应络合物的结构，以获得对TBPA简并识别的结构洞察。在底物及其类似物存在下进行的晶体浸泡实验获得的初步数据表明，在TBPA活性中心捕获低温反应中间体是可行的。为了明确区分低温捕获反应中间体的空间构型，我们计划收集至少2a分辨率的衍射数据。使用ADSC 3x3探测器在X25进行的数据收集使我们能够在天然晶体上以最高分辨率收集此类数据，否则由于我们晶体的晶胞尺寸较长，这是不可能的。

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. Class I glycolytic aldolases are enzymes that reversibly catalyze the cleavage of a biphosphorylated hexose, which yields glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP), after forming a protonated imine with a lysine residue. While the Tagatose-1,6-Bisphosphate Aldolase (TBPA) cleaves the tagatose-1,6-bisphosphate (TBP) and the fructose-1,6-bisphosphate (FBP) with similar affinities, the fructose-1,6-Bisphophate Aldolase (FBPA) is exclusively specific toward FBP. The cleavage/condensation products, differing only with regards as to stereochemical configuration of the third and fourth carbon atoms, raises the question of how aldolases stereospecifically recognize their cognate substrates. The rabbit muscle FBPA is well characterized, both kinetically and structurally. Moreover, an imine intermediate, trapped into the active site of the crystalline FBPA recently allowed the unveiling of significant aspects of the stereospecificity of this enzyme. However, the structural features of the permissive specificity of TBPA remain unknown. We have thus undertaken an x-ray crystallographic study of catalytic intermediates of the TBPA to reveal the molecular architecture of its active site and the residues involved in ligand recognition and catalysis. To this end, we have purified and crystallized native TBPA from Streptococcus pyogenes and its selenomethionylated derivative. MAD phasing experiments have allowed the determination of the tertiary structure of the enzyme revealing an (¿}¿¿/¿}¿¿)8 barrel subunit fold. We propose to determine the structures of reaction complexes formed with TBPA to high resolution to gain structural insight into the degenerate recognition by TBPA. Preliminary data obtained from crystal soaking experiemnts conducted in presence of substrate and their analogues show the feasibility of cryotrapping reaction intermediates in the TBPA active site. In order to unambiguously distinguish the steric configurations of cryotrapped reaction intermediates requires that we plan to collect diffraction data to at least 2A resolution. Data collection at X25 using the ADSC 3x3 detector has allowed us to collect such data to maximum resolution on native crystals otherwise not possible because of a long unit cell dimension in our crystals.