PROBING THE ACTIVE-SITE OF CYTOPLASMIC ALDEHYDE DEHYDROGENASE WITH A CHROMOPHORIC REPORTER GROUP

3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one ('DMNB') reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pK(a) of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD(+) to the modified enzyme, the pK(a) falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD(+). The modified enzyme binds NAD(+) very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD(+) when NADH is added.