Structural investigations on dehydrogenases by means of difference spectrophotometry

Abstract

The distribution and localization of tyrosine in glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde 3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) and in lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) were studied by difference spectrophotometry through spectrophotometric titration, solvent perturbation and iodination. In both dehydrogenases the alkaline dissociation of some phenolic hydroxyl groups is anomalous. The apparent pK′s are 11.3 and 11.5, and the titrations are irreversible. As measured by the solvent perturbation method, maxima of 45 and 50% of the tyrosyl residues may be located on the surfaces of the glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenases respectively. About 65 and 80% of the tyrosyl groups are accessible to iodine in glycine buffer (pH 9.2). The remainder becomes available to iodine only after the proteins have been denatured with concentrated urea. On the basis of the above findings the structural composition of these dehydrogenases is discussed.