Photoaffinity labeling of D-(-)-beta-hydroxybutyrate dehydrogenase by (arylazido)-beta-alanyl-substituted nicotinamide adenine dinucleotide.
(Arylazido)-beta-alanyl-substituted nicotinamide adenine dinucleotide (N3-NAD) is a photosensitive analogue of NAD capable of photoinduced nitrene generation and insertion into a nearby molecule. In the dark, N3-NAD can replace NAD as a cosubstrate for the mitochondrial D-(-)-beta-hydroxybutyrate dehydrogenase (BDH). With purified, phospholipid-reconstituted BDH and NAD as the variable substrate, the apparent Km and Vmax values were respectively 0.25 mM and 62.5 mumol min-1 (mg of protein)-1. With N3-NAD as the variable substrate, these values were respectively 0.59 mM and 5 mumol min-1 (mg of protein)-1. Photoirradiation of BDH in the presence of N3-NAD resulted in irreversible inhibition of the enzyme and incorporation into the protein of radioactivity from tritiated N3-NAD. Photoirradiation of BDH plus or minus NAD in the absence or presence of (arylazido)-beta-alanine caused little or no inhibition. The photoinhibition of BDH in the presence of N3-NAD was prevented nearly completely by addition of NADH, NAD plus beta-hydroxybutyrate, or NAD plus 2-methylmalonate and partially by addition of NAD. Moreover, the presence of NADH prevented, and prior partial modification of BDH at the NAD(H)-protectable site by N-ethylmaleimide decreased, the incorporation of radioactivity into BDH from photoirradiated [3H]N3-NAD. The above results suggest that N3-NAD can be used for photoaffinity labeling of BDH at the active site.
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D005033:Ethylmaleimide / Q000494:pharmacology; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014316:Tritium
D000345:Affinity Labels; D001386:Azides; C020606:arylazido-beta-alanyl-NAD; D009243:NAD; D014316:Tritium; D006884:Hydroxybutyrate Dehydrogenase; D005033:Ethylmaleimide