Betaine-aldehyde dehydrogenase from leaves of Amaranthus hypochondriacus L. exhibits an Iso Ordered Bi Bi steady state mechanism.
The kinetics of the oxidation of betaine aldehyde catalyzed by NAD(+)-betaine-aldehyde dehydrogenase, purified from amaranth leaves subjected to water deficit, were analyzed by steady state initial velocity and product and dead-end inhibition studies at low substrate concentrations. Only one product, NADH, gives inhibition. The other product of the reaction, glycine betaine, does not inhibit the enzyme even at concentrations as high as 10 mM. In dead-end inhibition experiments, AMP and choline were used as dead-end analogs of NAD+ and betaine aldehyde, respectively. The families of double-reciprocal plots in the range 0.010-0.500 mM NAD+ and 0.025-0.300 mM betaine aldehyde are linear and intersect at the left of the 1/v axis. NADH is a mixed inhibitor against NAD+ and betaine aldehyde. AMP is competitive with respect to NAD+ and mixed with betaine aldehyde. Choline is competitive against betaine aldehyde and uncompetitive with respect to NAD+. Our results are consistent with an Iso Ordered Bi Bi steady state mechanism in which NAD+ is the first substrate to bind to the enzyme and NADH is the last product to dissociate from it. To our knowledge, this is the first time that an Iso mechanism has been demonstrated by product inhibition studies, as predicted by Cleland (Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104-137).
Valenzuela-Soto
E M
EM
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autonoma de Mexico, México City.
Muñoz-Clares
R A
RA
D000249:Adenosine Monophosphate / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050728:Betaine-Aldehyde Dehydrogenase; D001667:Binding, Competitive; D002794:Choline / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010944:Plants / Q000201:enzymology*
D009243:NAD; D000249:Adenosine Monophosphate; D000445:Aldehyde Oxidoreductases; D050728:Betaine-Aldehyde Dehydrogenase; D002794:Choline