Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti conformation of NAD(+) nicotinamide but does not experience A-specific hydride transfer

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-depend ent oxidoreductase which catalyzes the oxidative phosphorylation of D-glyce raldehyde-3-phosphate (G3P) to form 1,3-diphosphoglycerate. The currently a ccepted mechanism involves an oxidoreduction step followed by a phosphoryla tion. 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 pyr i dinium si face requires optimal nicotinamidium-protein contacts for a sui table pyridinium-ring orientation. In previous studies carried out on Esche richia coil GAPDH (C. Corbier, A Mougin, Y. Mely, H. W. Adolph, M. Zeppezau er, D. Gerard, k Wonacott, and G. Branlant, Biochimie 72, 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 anc hor which favors the syn orientation of the nicotinamide ring, was examined . Here, we report further investigations on the molecular factors responsib le for the cofactor stereospecificity. Two single [Gly317] and [Ala317] GAP DH mutants and one double [Thr313- Gly317] GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of hol e GAPDW from E. coli (E. Duee, L. Olivier-Deyris, E. Fanchon, C. Corbier, G ;. Branlant, and O. Dideberg, J. Mel. Biol. 257, 814-838, 1996). The Kd con stants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAH are 5, 13, and 300 times higher than that of wild-type GAPDH. Transferred nucl ear Overhauser effect spectroscopy demonstrates that the wild-type syn orie ntation of bound nicotinamide remains unchanged in the [Gly317] and [Ala317 ] mutants, whereas a conformational equilibrium between the syn and anti fo rms occurs in the [Thr313-Gly317] double mutant with a preference for the a nti conformer. Although the double mutant preferably binds the nicotinamide ring in an anti conformation, it still exhibits B hydride transfer stereos pecificity. Yet, the catalytic efficiency is much less than that of the wil d type. This indicates that the hole GAPDH mutant fraction with an anti ori entation of bound NAD is not capable of forming the ternary complex with G3 P which would be required for an efficient A-specific catalytic process. Th e reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereo specificity of NAD(P)-dependent dehydrogenases, (C) 1999 Academic Press.