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 pKa 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 pKa 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.
Kitson
T M
TM
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Kitson
K E
KE
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D048588:Benzoxazines; D001665:Binding Sites; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000737:chemistry; D010078:Oxazines / Q000737:chemistry; D012756:Sheep
D048588:Benzoxazines; D010078:Oxazines; D009243:NAD; C087417:3,4-dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one; D000444:Aldehyde Dehydrogenase