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 dehy drogenase [D-glyceraldehyde-3-phosphate:NAD(+) oxidoreductase (phospho rylating), EC 1.2.1.12] from the host cytosolic fraction of soybean (G lycine nax L. Merr. cv. Williams) root nodules has been investigated b y steady-state initial velocity studies and inhibition studies with pr oducts and product analogs. The results were consistent with the enzym e having a bi-uni-uni-uni ping-pong mechanism in which NAD(+) and phos phate 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 e nzyme in a rapid-equilibrium fashion, whereas at pH 8.8 there was rapi d-equilibrium addition of both NAD(+) and phosphate to the enzyme. NAD H was a strong competitive inhibitor with respect to NAD(+) and phosph ate. The enzyme was very labile, especially above pH 8, but the rate o f 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. (C) 1994 Academic Press, Inc .