Gilboa R, Bauer A J, Shoham G
Department of Inorganic Chemistry and The Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Acta Crystallogr D Biol Crystallogr. 1998 Nov 1;54(Pt 6 Pt 2):1467-70. doi: 10.1107/s0907444997019720.
Two related and not thoroughly resolved issues in biochemistry concern the role, if any, of enzyme surfaces in routine metabolism and the method by which metabolic intermediates move between enzyme active sites during multi-step degradation or synthesis. An important enzyme for which a detailed three-dimensional structural analysis has been initiated is yeast glyceraldehyde 3-phosphate dehydrogenase (yGAP-DH). This enzyme is active as a tetramer of total molecular weight of 145 kDa and requires nicotinamide adenine dinucleotide (NAD+) as cofactor. In this report, the crystallization and preliminary crystallographic characterization of several crystal forms of yGAP-DH are described. Of the five distinct crystal forms, the most suitable was found to contain the holo-enzyme, and the crystals were grown by the vapor-diffusion method using polyethylene glycol 6000 as precipitant, sodium acetate as buffer (pH 4.6), and NAD+ and dithiothreitol as additives. The crystals belong to the orthorhombic space group P21212, with cell dimensions of a = 87.33, b = 96.11 and c = 115.34 A. These crystals are mechanically strong, relatively stable in the X-ray beam and diffract X-rays (from a normal rotating-anode radiation source) to better than 2 A resolution. A full 2.1 A resolution diffraction data set (98% completion) has been measured. The three-dimensional structures of related GAP-DH enzymes from several other sources have been determined and reported, and are available for a molecular replacement structure solution.
生物化学中有两个相关且尚未完全解决的问题，即酶表面在常规代谢中（若有）所起的作用，以及代谢中间体在多步降解或合成过程中在酶活性位点之间移动的方式。已开始对其进行详细三维结构分析的一种重要酶是酵母甘油醛-3-磷酸脱氢酶（yGAP-DH）。该酶以总分子量为145 kDa的四聚体形式具有活性，并且需要烟酰胺腺嘌呤二核苷酸（NAD +）作为辅因子。在本报告中，描述了yGAP-DH几种晶体形式的结晶及初步晶体学表征。在五种不同的晶体形式中，发现最合适的包含全酶，并且使用聚乙二醇6000作为沉淀剂、乙酸钠作为缓冲液（pH 4.6）以及NAD +和二硫苏糖醇作为添加剂，通过气相扩散法生长晶体。这些晶体属于正交晶系空间群P21212，晶胞尺寸为a = 87.33、b = 96.11和c = 115.34 Å。这些晶体机械强度高，在X射线束中相对稳定，并且能衍射X射线（来自常规旋转阳极辐射源）至优于2 Å的分辨率。已测量了完整的2.1 Å分辨率衍射数据集（完成度98%）。来自其他几种来源的相关GAP-DH酶的三维结构已被确定并报道，可用于分子置换结构解析。
