P-31 NUCLEAR-MAGNETIC-RESONANCE STUDIES ON COENZYME BINDING AND SPECIFICITY IN GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Binding of NAD(P)(+) to wild type and a series of mutants of the glyco lytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) f rom Bacillus stearothermophilus designed to alter the cofactor specifi city [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., & Branlant, G. (1993) Biochemistry 21, 10178-10184] has been studied by P-31 NMR. In the mutants with the L187A and P188S substitutions, the pyrophosphate signals are split, and the upfield resonance has been as signed to the P(a) phosphate. Titration of the NADP(+) 2'-phosphate pK (a) deduced from its chemical shift shows that the electrostatic envir onment in the binding site is largely affected by the single point mut ations. pK(a)s 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 ioniz ation state of its 2'-phosphate. In the quintuple mutant L33T-T34G-D35 G-L187A-P188S, designed in comparison with the photosynthetic NAD(P)-d ependent GAPDH of the chloroplast, the 2'-phosphate has a pK(a) of 6.8 . As further stabilizing interactions like hydrogen bonds or positivel y charged side chains would lower this pK(a), 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 D326-L187A-P188S are higher at p H 6.1 than at pH 8.1 and are similar at the same pH, indicating that t he difference in binding affinity between these three mutants results from the molecular recognition step or a conformational change upon bi nding.