Substrate inhibition by betaine aldehyde of betaine aldehyde dehydrogenase from leaves of Amaranthus hypochondriacus L.
We have previously proposed that at low substrate concentrations betaine aldehyde dehydrogenase follows an irreversible Iso Ordered Bi Bi Steady State kinetic mechanism with NAD+ as the leading substrate [E.M. Valenzuela-Soto and R.A. Muñoz-Clares, J. Biol. Chem. 268 (1993) 23818-23823]. To further the understanding of this enzyme, we have studied the kinetics at high substrate concentrations. Betaine aldehyde at concentrations above 500 microM behaves as a non-competitive inhibitor against NAD+, with downward-curved slope and intercept replots. Double-inhibition studies, using NADH as the second inhibitor, show the formation of the abortive ternary complex enzyme NADH betaine aldehyde, from which NADH may escape at a finite rate, accounting for the nonlinear Dixon plots obtained for both inhibitors. In addition, the binary complex enzyme x betaine aldehyde may give rise to a slower alternative route of reaction, which, under our experimental conditions, was observed at NAD+ concentrations above 1 mM, where double-reciprocal plots of initial velocity against [NAD+] and Dixon plots of 1/v against [NADH] were concave downward. In contrast with other aldehyde dehydrogenases, no 'substrate activation' by the aldehyde was observed under several conditions, which is consistent with the alternative route of reaction being slower than the route which operates at low substrate concentrations. Taken together, our results are consistent with the partial inhibition by high betaine aldehyde concentrations resulting from an irreversible Iso Random Steady State mechanism with a preferential route of reaction. Eventually, at very high betaine aldehyde concentrations, the kinetic mechanism may change to an apparent Ping Pong.
Vojtechová
M
M
Departamento de Bioquimica, Facultad de Química, Universidad Nacional Autónoma de México, México D.F.
Rodríguez-Sotres
R
R
Valenzuela-Soto
E M
EM
Muñoz-Clares
R A
RA
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001622:Betaine / Q000031:analogs & derivatives* / Q000494:pharmacology; D050728:Betaine-Aldehyde Dehydrogenase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D013696:Temperature
D009243:NAD; D001622:Betaine; C026820:betaine aldehyde; D000445:Aldehyde Oxidoreductases; D050728:Betaine-Aldehyde Dehydrogenase