Linear free energy relationship studies were carried out to examine the nature of the interaction of aldehydes with glyceraldehyde-3-phosphate dehydrogenase. The association constant of most aldehydes with the enzyme is dependent on the Taft polar substituent constant, characterized by a p* value of 1.7. This is identical with the p* value for thiohemiacetal formation between aldehydes and glutathione and suggests that the binding of aldehydes to the enzyme results in the formation of a thiohemiacetal adduct. d-Glyceraldehyde 3-phosphate, however, shows a positive deviation from the linear correlation, indicating a specific interaction of the phosphate substituent with the enzyme. Thiohemiacetal formation contributes 3.9 kcal/mol to the formation of the enzyme-aldehyde complex with d-glyceraldehyde 3-phosphate, and the interaction of the phosphate moiety on the aldehyde with the phosphate binding site on the enzyme contributes 3.7 kcal/mol. The interaction of inorganic phosphate with the aldehyde phosphate binding site on the enzyme is characterized by a ΔG° > -1.4 kcal/mol, indicating that most of the apparent free energy for binding the phosphate substituent on the aldehyde is entropic in origin. Phosphorylation of irreversible inhibitors of glyceralde-hyde-3-phosphate dehydrogenase has been shown to enhance their reactivity with the enzyme by 3.8 (±1) kcal/mol [Byers, L. D. (1977) J. Am. Chem. Soc. 99, 4146-4149], Phosphorylation of the aldehyde substrates also enhances the stereoselectivity of the overall oxidative reaction [Byers, L. D. (1978) Arch. Biochem. Biophys. 186, 335-342], In addition to these effects, and the enhanced binding of aldehydes, the phosphate substituent was found to alter the conformational mobility of the protein, the specificity of deacylation of the acyl enzyme (with respect to acyl acceptors), and the reactivity of the aldehydes as substrates in the overall oxidative arsenolysis reaction. On the basis of electronic effects of the acyl substituent, d-glyceraldehyde 3-phosphate is more reactive than are nonphosphorylated aldehydes by a factor of 104. © 1979, American Chemical Society. All rights reserved.
