Photoaffinity labeling of lactate dehydrogenase by the carbene derived from the 3-diazirino analog of nicotinamide adenine dinucleotide

Publisher Website
Google Scholar
Abstract
The 3-diazirino analog of NAD+, DAD+ [3-(3H-diazirino)pyridine adenine dinucleotide], in which the diazirine group replaces the carboxamide in an almost isosteric substitution, was 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, 3 types of interaction can be discerned and separately quantitated 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; and 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 .apprx. 0.3. 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+ analog.