Kinetic properties of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from the host fraction of soybean root nodules.

The kinetic mechanism of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] from the host cytosolic fraction of soybean (Glycine max L. Merr. cv. Williams) root nodules has been investigated by steady-state initial velocity studies and inhibition studies with products and product analogs. The results were consistent with the enzyme having a bi-uni-uni-uni ping-pong mechanism in which NAD+ and phosphate interacted sequentially with the enzyme, followed in turn by the release of 1,3-bisphosphoglycerate, the addition of D-glyceraldehyde-3-phosphate, and the release of NADH. At pH 7.2, NAD+ bound to the enzyme in a rapid-equilibrium fashion, whereas at pH 8.8 there was rapid-equilibrium addition of both NAD+ and phosphate to the enzyme. NADH was a strong competitive inhibitor with respect to NAD+ and phosphate. The enzyme was very labile, especially above pH 8, but the rate of loss of activity was considerably reduced in the presence of NAD+ and phosphate. The possible role of these properties in the regulation of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in the host cytosol of soybean nodules is discussed.