Phosphate-binding sites in phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

The two anion-binding sites of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GraP-DH), the Ps and Pi sites, were originally proposed by Moras et al. [Moras, D., Olsen, K.W., Sabesan, M.N., Buehner, M., Ford, G.C. & Rossmann, M. G. (1975) J. Biol. Chem. 250, 9137-9162] to bind the C3 phosphate of the glyceraldehyde 3-phosphate and the inorganic phosphate respectively. Ps site mutants T179A, and T179M, and R231L, and the Pi site mutants T150A and T208 of the Bacillus stearothermophilus GraP-DH were constructed by site-directed mutagenesis and their kinetic properties were determined and compared with those of mutants R195L and R231G, already described [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260-3265]. Taking advantage of the opportunity to study both the oxidoreduction and the phosphorylation step independently and the fact that the phosphorylation becomes rate determining for most of the mutants, the relative energetic contribution of each mutated amino acid to the phosphorylation step was evaluated. It was concluded that (a) Ps amino acids contribute more than the Pi amino acids to the stabilisation of the transition state relative to the ground state and (b) the side chain of arginine contributes more than that of the threonine residue. It was also concluded that the differences observed in the efficiency of the phosphorylation step for Ps and Pi mutants is a consequence of the orientation of the thioester bond of the thioacyl] intermediate relative to the attacking inorganic phosphate and not of a change in the intrinsic electrophilic property of the thioacyl intermediate. Furthermore, the kinetic results on the overall steps leading to the acyl-enzyme formation provided supplementary evidence that the C3 phosphate moiety of the glyceraldehyde 3-phosphate interacts with the Pi site during these steps and thus are consistent with the findings of Skarzynski et al. [Skarzynski, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol Biol. 193, 171-183] and Corbier et al. [Corbier, C., Michels, S., Wonacott, A. & Branlant, G. (1994) Biochemistry 33, 3260-3265] that recommended the reconsideration of the first definition of the Ps and Pi sites.