The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 22.214.171.124) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear ‘mixed inhibition’ pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.
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Research Article| October 01 1992
Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes
G T M Henehan;
Biochem J (1992) 287 (1): 145–150.
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G T M Henehan, K F Tipton; Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes. Biochem J 1 October 1992; 287 (1): 145–150. doi: https://doi.org/10.1042/bj2870145
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