Dimers generated from tetrameric phosphorylating glyeceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus are inactive but exhibit cooperativity in NAD binding

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been described as a "dimer of dimers" with three nonequivalent interfaces, P-axis (between subunits O and P and between subunits Q and R), Q-axis (between subunits O and R and between sub units P and R), and R-axis interface (between subunits O and R and between subunits P and Q). O-P dimers, the most stable and the easiest to generate, have been created by selective disruption of hydrogen bonds across the R- and Q-axis interfaces by site-directed mutagenesis. Asp-186 and Ser-48, and Glu-276 and Tyr-46, which are hydrogen bond partners across the X- and e-a xis interfaces, respectively, have been replaced with glycine residues. All mutated residues are highly conserved among GAPDHs from different species and are located in loops. Both double mutants D186G/E276G and Y46G/S48G wer e dimeric, while all single mutants remained tetrameric. As previously desc ribed [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., and B ranlant, C. (1993) Biochemistry 32, 10178-10184], NAD binding to wild type GAPDH (wtGAPDH) was interpreted according to the induced-fit model and exhi bited negative cooperativity. However, NAD binding to wtGAPDH can be adequa tely described in terms of two independent dimers with two interacting bind ing sites in each dimer. Single mutants D186G, E276G, and Y46G exhibited be havior in NAD binding similar to that of the wild type, while both dimeric mutants D186G/E276G and Y46G/S48G exhibited positive cooperativity in bindi ng the coenzyme NAD. The fact that O-P dimer mutants retained cooperative b ehavior shows that (1) the P-axis interface is important in transmitting th e information induced upon NAD binding inside the O-P dimer from one subuni t to the other and (2) the S-loop of the R-axis-related subunit is not dire ctly involved in cooperative binding of NAD in the O-P dimer. In both O-P d imer mutants, the absorption band of the binary enzyme-NAD complex had a hi ghly decreased intensity compared to that of the wild type and, in addition , totally disappeared in the presence of G3P or 1,3-dPG. However, no enzyma tic activity was detected, indicating that the formed ternary enzyme-NAD-G3 P or -1,3-dPG complex was not catalytically efficient. In the O-P dimers, t he interaction with the S-loop of the R-axis-related subunit is disrupted, and therefore, the S-loop should be less structured. This resulted in incre ased accessibility of the active site to the solvent, particularly for the adenosine-binding site of NAD. Thus, together, this is likely to explain bo th the lowered affinity of the dimeric enzyme for NAD and the absence of ac tivity.