INTERACTION OF A SPIN-LABELED ANALOG OF NICOTINAMIDE-ADENINE DINUCLEOTIDE WITH ALCOHOL DEHYDROGENASE .I. SYNTHESIS KINETICS AND ELECTRON PARAMAGNETIC RESONANCE STUDIES

An analog of nicotinamide-adenine dinucleotide containing an unpaired electron has been prepared and shown to inhibit liver alcohol dehydrogenase competitively with respect to nicotinamide-adenine dinucleotide (= 5 ± 2 μM). The compound, adenosine 5′-diphosphate-4(2,2,6,6,-tetramethylpiperidine-1-oxyl), was formed by coupling adenosine monophosphate to 2,2,6,6-tetramethyl-4-phosphopiperidine-1-oxyl. The electron paramagnetic resonance of the spin-labeled compound broadened when bound to liver alcohol dehydrogenase. Titrating the enzyme with the spin-labeled analog and measuring the decrease in amplitude of the electron paramagnetic resonance spectrum revealed that the enzyme possessed two classes of binding sites: two sites which bind the analog with a= 17 ± 8μM, in agreement with its, and five to six sites which bind with a = 75 ± 9μM. Reduced nicotinamide-adenine dinucleotide can only displace the bound radical from the strong binding sites, not from the five or six weak binding sites. Zinc-free liver alcohol dehydrogenase has been prepared and was shown to possess the two strong binding sites for the spin-labeled analog with a KD = 27 ± 6μM. The apoenzyme does not possess the weak binding sites. The analog can be displaced by reduced nicotinamide-adenine dinucleotide, showing that the enzymatically inactive apoenzyme can still bind coenzyme. © 1969, American Chemical Society. All rights reserved.