A substrate in pieces: allosteric activation of glycerol 3-phosphate dehydrogenase (NAD+) by phosphite dianion.
The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD (+), GPDH) saturated with NADH is (1.0 x 10 (6) M (-1) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 1.1 x 10 (8), which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH ( K d > 0.1 M), but the bound dianion strongly activates GLY toward enzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M (-2) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 5 x 10 (5) M (-1), which was used to calculate an intrinsic phosphite binding energy of -7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structural data provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring (1) a binding pocket complementary to the reactive substrate fragment which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.
Tsang
Wing-Yin
WY
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA.
Amyes
Tina L
TL
Richard
John P
JP
D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D000838:Anions / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D017905:Phosphites / Q000737:chemistry / Q000494:pharmacology*; D011817:Rabbits; D013379:Substrate Specificity
D000838:Anions; D017905:Phosphites; D005993:Glycerolphosphate Dehydrogenase