Structural analysis of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli: Direct evidence of substrate binding and cofactor-induced conformational changes

The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) f rom Escherichia coli have been determined in three different enzymatic stat es, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor bin ding are limited to the residues that bind the adenine moiety of NAD(+). Gl yceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P-i" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with th e flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorpo ration of the new P-i site in this model, it is now proposed that the C3 ph osphate of GAP initially binds at the new P-i site and then flips to the P- i site before hydride transfer. A superposition of NAD(+)-bound and hemiace tal intermediate structures reveals an interaction between the hydroxyl oxy gen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding sug gests chat the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP bi nding.