Photoaffinity labeling of lactate dehydrogenase by the carbene derived from the 3-diazirino analogue of nicotinamide adenine dinucleotide.
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.
D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives*; D010782:Photolysis; D011485:Protein Binding; D011817:Rabbits
D000345:Affinity Labels; D009243:NAD; D007770:L-Lactate Dehydrogenase