Crystallization and preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from Saccharomyces cerevisiae (baker's yeast)

Two related and not thoroughly resolved issues in biochemistry concern the role, if any, of enzyme surfaces in routine metabolism and the method by wh ich metabolic intermediates move between enzyme active sites during multi-s tep degradation or synthesis. An important enzyme for which a detailed thre e-dimensional structural analysis has been initiated is yeast glyceraldehyd e 3-phosphate dehydrogenase (yGAP-DH). This enzyme is active as a tetramer of total molecular weight of 145 kDa and requires nicotinamide adenine dinu cleotide (NAD(+)) as cofactor. In this report, the crystallization and prel iminary 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 ace tate as buffer (pH 4.6), and NAD(+) and dithiothreitol as additives. The cr ystals belong to the orthorhombic space group P2(1)2(1)2, with cell dimensi ons of a = 87.33, b = 96.11 and c - 115.34 A. These crystals are mechanical ly strong, relatively stable in the X-ray beam and diffract X-rays (from a normal rotating-anode radiation source) to better than 2 Angstrom resolutio n. A full 2.1 Angstrom 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 avail able for a molecular replacement structure solution.