Coenzyme-induced changes in the optical rotatory dispersion properties of glyceraldehyde 3-phosphate dehydrogenase.

The high binding affinity of diphosphopyridine nucleotide for glyceraldehyde S-phosphate dehydrogenase was first estimated quantitatively by Velick, Hayes, and Harting (1) by using an ultracentrifugation separation method. A dissociation constant of the order of 10-7 M (2) with a stoichiometry of 3 to 4 moles of DPN+ per mole of enzyme was determined (3). Several characteristics of the apoenzyme differ from those of the holoenzyme. The apoenzyme exhibits a greater thermal lability than the native enzyme (4), has resisted crystallization (5) (except in an acylated form (3)), and is more susceptible to proteolysis (6). Also, Boyer and Schulz (7) have demonstrated an increase in negative specific rotation after removal of DPN+. Optical rotatory dispersion techniques have been used extensively for the determination of protein structure (8, 9). Recently, similar techniques have been used to study cofactorprotein interactions (10-12). Thus, for liver alcohol dehydrogenase, Ulmer, Li, and Vallee (13) described an extrinsic Cotton effect at 327 rnp that was dependent upon binding of reduced pyridine nucleotide. Modifications of optical rotatory properties in the presence of specific inhibitors (14, 15), metal chelators (la), and coenzyme analogues (17) also have been investigated. The indications already cited that coenzyme-dependent changes occur in the properties of glyceraldehyde S-phosphate dehydrogenase suggested that changes in optical rotatory dispersion properties may also occur. In this study we report that, unlike the case with liver alcohol dehydrogenase, no extrinsic Cotton effect in the 330 rnp region was produced by DPNH binding to glyceraldehyde Y-phosphate dehydrogenase. However, other effects related to coenzyme binding were observed, including large changes in parameters of rotatory dispersion and variance in magnitude of an inflection point in the 280 rnp spectral region.