STRUCTURAL STUDIES ON SUBSTRATE AND INHIBITOR COMPLEXES OF CHICKEN LDH

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。鸡胸肌乳酸脱氢酶（LDH）是碳水化合物代谢的必需酶。虽然同源同源物的氨基酸数据和原子配位结构信息是可用的，但鸡肌肉LDH-A的结构尚未确定。从鸡胸肌中分离纯化了LDH-A。对土鸡LDH-A和各种底物、辅因子和抑制剂络合物进行结晶。将天然蛋白晶体以及丙酮酸、NAD、NADH和草酸盐配合物快速冷却后运至SSRL，在BL9-1和BL9-2光束线上收集衍射数据。一些数据是作为大分子晶体学课程的一部分，从富勒顿州立大学远程收集的。以猪LDH-A为搜索模型，采用分子置换的方法对土鸡结构（a=84.04¿¿b=126.78¿¿c=252.74¿¿）进行了1.9°分辨率的求解。以土鸡结构为搜索模型，在不对称单元中有一个四聚体，采用分子置换法求解了LDH-A与丙酮酸配合物的结构（空间群C2， a=75.16¿¿b=152.40¿¿c=142.79¿¿beta=93.5¿）至1.9´´分辨率。结构的确定和细化已经开始使用2.3¿¿和1.9¿¿数据的LDH-A与NAD和NADH络合。在乳酸盐和草酸盐的配合下，生长出了新的鸡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.