Standring D N, Knowles J R
Biochemistry. 1980 Jun 10;19(12):2811-6. doi: 10.1021/bi00553a042.
The 3-diazirino analogue of NAD+, DAD+, in which the diazirine group replaces the carboxamide in an almost isosteric substitution, has been synthesized as a photoaffinity reagent for dehydrogenases. With lactate dehydrogenase, the Kdiss is 4 mM (compare the NAD+ Kdiss of 0.6 mM). On photolysis, three types of interaction can be discerned and separately quantitiated by using [3H]DAD+: noncovalent binding of the photoproducts of DAD+, which is removed by protein denaturation; nonspecific covalent labeling, which is eliminated by the presence of a scavenger such as glutathione; specific covalent labeling at the active site, which is prevented by competition from the natural ligands. The photolabeling efficiency (sites covalently labeled/sites initially occupied) is approximately 0.3. It is evident that the carbene generated from DAD+ is more effective than that from the more bulky 3-diazoacetate or than the less reactive nitrene derived from the 3-azido-NAD+ analogue.
NAD+的3-重氮丙啶类似物DAD+，其中重氮丙啶基团取代了羧酰胺，几乎是等排取代，已被合成为脱氢酶的光亲和试剂。对于乳酸脱氢酶，解离常数Kdiss为4 mM（相比之下，NAD+的Kdiss为0.6 mM）。光解时，通过使用[3H]DAD+可识别并分别定量三种相互作用：DAD+光产物的非共价结合，可通过蛋白质变性去除；非特异性共价标记，可通过存在诸如谷胱甘肽的清除剂消除；活性位点的特异性共价标记，可通过天然配体的竞争阻止。光标记效率（共价标记的位点/最初占据的位点）约为0.3。很明显，由DAD+产生的卡宾比由体积更大的3-重氮乙酸酯产生的卡宾或由3-叠氮基-NAD+类似物衍生的活性较低的氮烯更有效。
