Complexes of NADH with betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus L

The kinetic mechanism of betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus is ordered with NAD(+) adding first. NADH is a noncompetitive inhibitor against NAD(+), which was interpreted before as evidence of an iso mechanism, in which NAD(+) and NADH binds to different forms of free enzyme. With the aim of testing the proposed kinetic mechanism, we have now investigated the ability of NADH to form different complexes with the enzyme. By initial velocity and equilibrium binding studies, we found that the steady-state levels of E.glycine betaine are negligible, ruling out binding of NADH to this complex. However, NADH readily bind to E.betaine aldehyde, whose levels most likely are kinetically significant given its low dissociation constant. Also, NADH combined with E.NADH and E.NAD(+). Finally, NADH was not able to revert the hydride transfer step, what suggest that there is no acyl-enzyme intermediate, i.e. the release of the reduced dinucleotide takes place after the deacylation step. Although formation of the complex E.NAD(+).NADH would produce an uncompetitive effect in the inhibition of NADH against NAD(+), the iso mechanism cannot be conclusively discarded.