Kitson T M, Kitson K E
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Biochem J. 1994 May 15;300 ( Pt 1)(Pt 1):25-30. doi: 10.1042/bj3000025.
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.
3,4-二氢-3-甲基-6-硝基-2H-1,3-苯并恶嗪-2-酮（“DMNB”）与细胞质醛脱氢酶的反应方式，与之前观察到的结构相关的对硝基苯基二甲基氨基甲酸酯类似，但在酶的活性位点提供了一个共价连接的含对硝基苯酚的报告基团。酶连接的报告基团的pKa远高于游离对硝基苯酚的pKa，这与其处于非常疏水的环境或可能含有负电荷的环境一致。当NAD⁺与修饰后的酶结合时，pKa急剧下降，下降约4.5个pH单位。这意味着在这些条件下，对硝基苯氧离子部分附近存在正电荷，可能是NAD⁺烟酰胺环的正电荷。修饰后的酶与NAD⁺结合非常紧密；凝胶过滤和透析都无法有效分离它们。然而，报告基团提供了一种方便的方法，用于监测添加NADH时结合的NAD⁺的置换情况。
