Reaction between Sheep Liver Mitochondrial Aldehyde Dehydrogenase and A Chromogenic ‘Reporter Group’ Reagent

Abstract

The cytosolic form of aldehyde dehydrogenase (ALDH-1) has been shown to react slowly with p-nitrophenyl dimethylcarbamate, liberating p-nitrophenoxide and giving an inactive from the enzyme in which Cys-302 carries a -CO-NMe₂ label (Kitson et al., 1991) is work led to the idea that a cyclic analogue of the carbamate would constitute a ‘eporter group’ reagent of the type originally envisaged by Burr and Koshland (1964), since the chromophoric p-nitrophenoxide moiety would end up covalently bound within the ene’s active site. The compound in question, namely 3,4-dihydro-3-methyl-6-ni- tro-2-l,3-benzoxazin-2-one or DMNB (see Figure 1) was synthesised and shown to react in the expected way with esterases such such as chymotrypsin (Kitson and Freeman, 1993). Furtheork with ALDH-1 showed that in this case the pK_a of the p-nitrophenol reporter group perturbed upwards by about 3 pH units (Kitson and Kitson, 1994). This rather dramatic observation was interpreted to mean that the substrate binding site of ALDH-1 is either very hydrophobic character or contains a negatively charged amino acid sidechain (or conceivably has both characteristics).