Covalent Modification of Sheep Liver Cytosolic Aldehyde Dehydrogenase by the Oxidative Addition of Coloured Phenoxazine, Phenothiazine and Phenazine Derivatives

Abstract

Aldehyde dehydrogenase acts as an esterase towards reactive esters such as p-nitrophenyl acetate, and although the subject of prolonged debate, it is now generally believed that the dehydrogenase and esterase actions of the enzyme involve the same active site and catalytic groups (see Kitson and Kitson, 1996, and references therein). With p -nitrophenyl dimethylcarbamate, the reactivity (compared to the acetate) is so drastically reduced that it takes several hours for this ‘substrate’ to acylate cytosolic aldehyde dehydrogenase (ALDH-l), and the rate of subsequent deacylation is essentially zero (Kitson et al., 1991). Thus the carbamate acts as an active-site-directed irreversible inactivator of the enzyme. It was thought that resorufin dimethylcarbamate (see Figure 1) would react likewise, but since the molar absorptivity of the resorufin anion (69,700) is so much greater than that of p-nitrophenoxide (18,320) (Kitson, 1996), the resorufin carbamate would be a much more sensitive active site titrant than the p-nitrophenyl compound. The results reported and discussed below show that this expectation was not borne out. Resorufin dimethylcarbamate does inactivate ALDH-I, but the chemistry of the reaction is more complicated and interesting than the simple acylation process expected from the previous work with the p-nitrophenyl equivalent; it may be termed ‘oxidative addition’.