SUBSTRATE-INHIBITION BY BETAINE ALDEHYDE OF BETAINE ALDEHYDE DEHYDROGENASE FROM LEAVES OF AMARANTHUS-HYPOCHONDRIACUS L

We have previously proposed that at low substrate concentrations betai ne aldehyde dehydrogenase follows an irreversible Iso Ordered Bi Bi St eady State kinetic mechanism with NAD(+) as the leading substrate [E.M . Valenzuela-Soto and R.A. Munoz-Clares, J. Biol. Chem. 268 (1993) 238 18-23823]. To further the understanding of this enzyme, we have studie d the kinetics at high substrate concentrations. Betaine aldehyde at c oncentrations above 500 mu M behaves as a non-competitive inhibitor ag ainst NAD(+), with downward-curved slope and intercept replots. Double -inhibition studies, using NADH as the second inhibitor, show the form ation of the abortive ternary complex enzyme NADH betaine aldehyde, fr om which NADH may escape at a finite rate, accounting for the nonlinea r Dixon plots obtained for both inhibitors. In addition, the binary co mplex enzyme betaine aldehyde may give rise to a slower alternative ro ute of reaction, which, under our experimental conditions, was observe d at NAD(+) concentrations above: 1 mM, where double-reciprocal plots of initial velocity against [NAD(+)] and Dixon plots of l/v against [N ADH] were concave downward. In contrast with other aldehyde dehydrogen ases, no 'substrate activation' by the aldehyde was observed under sev eral conditions, which is consistent with the alternative route of rea ction being slower than the route which operates at low substrate conc entrations. Taken together, our results are consistent with the partia l inhibition by high betaine aldehyde concentrations resulting from an irreversible Iso Random Steady State mechanism with a preferential ro ute of reaction. Eventually, at very high betaine aldehyde concentrati ons, the kinetic mechanism may change to an apparent Ping Pong. (C) 19 97 Elsevier Science B.V.