The binding of NADH to cytoplasmic aldehyde dehydrogenase after modification with p-nitrophenyl dimethylcarbamate.

Cytoplasmic aldehyde dehydrogenase may be modified by reaction with p-nitrophenyl dimethylcarbamate to give a stable E-X-CO-NMe2 species that is an analogue of the usual labile acyl-enzyme involved in the enzyme's reactions. [X is derived from an enzymic nucleophilic group.] This species still contains the tightly bound NADH that is present in the native enzyme. When further NADH binds to E-X-CO-NMe2 its fluorescence is enhanced over 4 times more than when it binds to unmodified enzyme; this fluorescence is completely unaffected by high propionaldehyde concentration and only slightly affected by p-nitrobenzaldehyde. The modified species has 1.0 NADH binding site in the absence of Mg2+ and 1.67 sites in its presence. The rate of dissociation of E-X-CO-NMe2.NADH is biphasic (k 3.4 and 1.8 min-1) and is considerably lower than that of E.NADH; the presence of Mg2+ slows the process even more (k 0.47 and 0.37 min-1). The implications of these studies towards a greater understanding of the nature of aldehyde dehydrogenase-catalysed reactions are discussed.