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

被引:79
作者
WEINER, H
机构
[1] Department of Biochemistry, Purdue University, Lafayette
关键词
D O I
10.1021/bi00830a011
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:526 / &
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