STRUCTURAL STUDIES ON SUBSTRATE AND INHIBITOR COMPLEXES OF CHICKEN LDH

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。鸡胸肌乳酸脱氢酶(LDH)是碳水化合物代谢的重要酶。虽然已有同源同源物的氨基酸数据和原子配位结构信息，但鸡肌肉LDH-A的结构尚未确定。从鸡胸肌中分离纯化了乳酸脱氢酶A。将天然鸡肉LDH-A与各种底物、辅因子和抑制剂络合物进行了结晶。天然蛋白质的晶体以及丙酮酸、NAD、NADH和草酸络合物被闪冷并运送到SSRL，在那里收集了光束线BL9-1和BL9-2的衍射数据。一些数据是从加州大学富勒顿分校远程收集的，作为大分子结晶学课程的一部分。以猪乳酸脱氢酶-A为搜索模型，通过分子置换，将天然鸡肉的结构(a=84.04b=126.78？c=252.74？)解析为1.9？丙酮酸与乳酸脱氢酶A的配位结构(C2空间群，a=75.16b=152.40，c=142.79β=93.5)，以天然鸡的结构为搜索模型，用分子置换的方法得到了1.9°的分辨率，其中一个四聚体位于不对称单元中。LDH-A分别与NAD和NADH络合后，已开始使用2.3和1.9数据进行结构测定和精炼。在乳酸盐和草酸盐的络合物中，生长了新的鸡肉LDH-A晶体。

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. Lactate dehydrogenase (LDH) from chicken breast muscle is an essential enzyme in carbohydrate metabolism. Although amino acid data and atomic coordinate structure information are available for orthologous homologs, the structure of chicken muscle LDH-A had not yet been determined. LDH-A from chicken breast muscle was isolated and purified. Native chicken LDH-A and various substrate, cofactor and inhibitor complexes were crystallized. Crystals of native protein, as well as pyruvate, NAD, NADH and oxalate complexes, were flash cooled and shipped to SSRL, where diffraction data were collected on beam lines BL9-1 and BL9-2. Some data was collected remotely from CSU Fullerton as part of a Macromolecular Crystallography course. The native chicken structure (a=84.04 ¿¿ b=126.78 ¿¿ c=252.74 ¿¿ has been solved to 1.9 ¿¿resolution by molecular replacement, using porcine LDH-A as the search model. The structure of LDH-A complexed with pyruvate (space group C2, a=75.16 ¿¿ b=152.40 ¿¿ c=142.79 ¿¿ beta=93.5¿¿) has been also been solved to 1.9 ¿¿resolution by molecular replacement, using the native chicken structure as the search model, with one tetramer in the asymmetric unit. Structure determination and refinement have begun using 2.3 ¿¿and 1.9 ¿¿data for LDH-A complexed with NAD and NADH respectively. New crystals of chicken LDH-A, in complex with lactate and oxalate, have been grown.