PHOSPHATE-BINDING SITES IN PHOSPHORYLATING GLYCERALDEHYDE-3-PHOSPHATEDEHYDROGENASE FROM BACILLUS-STEAROTHERMOPHILUS

The two anion-binding sites of the glycolytic glyceraldehyde-3-phospha te dehydrogenase (GraP-DH), the Ps and Pi sites, were originally propo sed 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, 9137-916 2] to bind the C3 phosphate of the glyceraldehyde 3-phosphate and the inorganic phosphate respectively. Ps site mutants T179A, and T179M, an d R231L, and the Pi site mutants T150A and T208A of the Bacillus stear othermophilus GraP-DH were constructed by site-directed mutagenesis an d their kinetic properties were deter mined and compared with those of mutants R195L and R231G, already described [Corbier, C., Michels, S., Wonacott, A. Br Branlant, G. (1994) Biochemistry 33, 3260-3265]. Taki ng advantage of the opportunity to study both the oxidoreduction and t he phosphorylation step independently and the fact that the phosphoryl ation becomes rate determining for most of the mutants, the relative e nergetic contribution of each mutated amino acid to the phosphorylatio n step was evaluated. It was concluded that (a) Ps amino acids contrib ute more than the Pi amino acids to the stabilisation of the transitio n state relative to the ground state and (b) the side chain of arginin e contributes more than that of the threonine residue. It was also con cluded that the differences observed in the efficiency of the phosphor ylation step for Ps and Pi mutants is a consequence of the orientation of the thioester bond of the thioacyl intermediate relative to the at tacking inorganic phosphate and not of a change in the intrinsic elect rophilic property of the thioacyl intermediate. Furthermore, the kinet ic results on the overall steps leading to the acyl-enzyme formation p rovided supplementary evidence that the C3 phosphate moiety of the gly ceraldehyde 3-phosphate interacts with the Pi site during these steps and thus are consistent with the findings of Skarzynski et al. [Skarzy nski, T., Moody, P C. E. & Wonacott, A. J. (1987) J. Mel. Biol. 193, 1 71-183] and Corbier et al. [Corbier, C., Michels, S., Wonacott, A. st Branlant, G. (1994) Biochemistry 33, 3260-3265] that recommended the r econsideration of the first definition of the Ps and Pi sites.