Engineered Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Binds theAntiConformation of NAD+Nicotinamide but Does Not Experience A-Specific Hydride Transfer

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent oxidoreductase which catalyzes the oxidative phosphorylation ofd-glyceraldehyde-3-phosphate (G3P) to form 1,3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. GAPDH is classified as a B-specific oxidoreductase. The inspection of several crystal structures of GAPDHs indicates that the efficient hydride transfer from the hemithioacetal intermediate to the C4 position of the pyridiniumsiface requires optimal nicotinamidium–protein contacts for a suitable pyridinium-ring orientation. In previous studies carried out onEscherichia coliGAPDH (C. Corbier, A. Mougin, Y. Mely, H. W. Adolph, M. Zeppezauer, D. Gerard, A. Wonacott, and G. Branlant,Biochimie72, 545–554, 1990; J. Eyschen, C. Corbier, B. Vitoux, G. Branlant, and M. T. Cung,Protein Pept. Lett.1, 19–24, 1994), the role of the invariant Asn 313 residue, as an anchor which favors thesynorientation of the nicotinamide ring, was examined. Here, we report further investigations on the molecular factors responsible for the cofactor stereospecificity. Two single [Gly317] and [Ala317] GAPDH mutants and one double [Thr313-Gly317] GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of holo GAPDH fromE. coli(E. Duée, L. Olivier-Deyris, E. Fanchon, C. Corbier, G. Branlant, and O. Dideberg,J. Mol. Biol.257, 814–838, 1996). TheKdconstants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAD are 5, 13, and 300 times higher than that of wild-type GAPDH. Transferred nuclear Overhauser effect spectroscopy demonstrates that the wild-typesynorientation of bound nicotinamide remains unchanged in the [Gly317] and [Ala317] mutants, whereas a conformational equilibrium between thesynandantiforms occurs in the [Thr313-Gly317] double mutant with a preference for theanticonformer. Although the double mutant preferably binds the nicotinamide ring in ananticonformation, it still exhibits B hydride transfer stereospecificity. Yet, the catalytic efficiency is much less than that of the wild type. This indicates that the holo GAPDH mutant fraction with anantiorientation of bound NAD is not capable of forming the ternary complex with G3P which would be required for an efficient A-specific catalytic process. The reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases.