Binding of NAD(P)+ to wild type and a series of mutants of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus designed to alter the cofactor specificity [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., & Branlant, G. (1993) Biochemistry 21, 10178-10184] has been studied by 31P NMR. In the mutants with the L187A and P188S substitutions, the pyrophosphate signals are split, and the upfield resonance has been assigned to the P(a) phosphate. Titration of the NADP+ 2'-phosphate pKa deduced from its chemical shift shows that the electrostatic environment in the binding site is largely affected by the single point mutations. pKas ranging from 7.7 for the L187A-P188S mutant to < 5.7 for the D32G-L187A-P188S and D32A-L187A-P188S mutants have been observed, thus indicating that the binding of NADP+ is modulated by the ionization state of its 2'-phosphate. In the quintuple mutant L33T-T34G-D35G-L187A-P188S, designed in comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, the 2'-phosphate has a pKa of 6.8. As further stabilizing interactions like hydrogen bonds or positively charged side chains would lower this pKa, it is suggested that the 2'-phosphate ionization state of bound NADP+ in chloroplastic GAPDH is dianionic. The NADP+ dissociation rate constants (k(off)) of the three mutants D32G, L187A-P188S, and D32G-L187A-P188S, are higher at pH 6.1 than at pH 8.1 and are similar at the same pH, indicating that the difference in binding affinity between these three mutants results from the molecular recognition step or conformational change upon binding.