The role of nicotinamide adenine dinucleotide in the inhibition of bovine liver S-adenosylhomocysteine hydrolase by neplanocin A.
Neoplanocin A, a cyclopentenyl analog of adenosine, has been shown recently to be a tight binding inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), exhibiting a stoichiometry of one molecule of inhibitor per molecule of the enzyme tetramer (Borchardt, R. T., Keller, B. T., and Patel-Thombre, U. (1984) J. Biol. Chem. 259, 4353-4358). In the present study a detailed analysis was performed of the possible role of the enzyme-bound NAD+ in the inactivation of AdoHcy hydrolase by neplanocin A. The NAD+/NADH content was quantitated using a fluorescence technique. The native enzyme showed intrinsic fluorescence with an emission maximum at 460 nm when excited at 340 nm, partially due to NADH bound to the enzyme. It was found that the content of NAD+ and NADH in freshly prepared, native enzyme is equal, having a stoichiometry of two nucleotides per enzyme molecule (tetramer). In addition, it was observed that the enzymatic activity of the native enzyme can be increased by about 30% following preincubation with NAD+. Furthermore, it was demonstrated that the mechanism of inhibition of AdoHcy hydrolase by neplanocin A involves the reduction of enzymatically bound NAD+ to NADH. Catalytic activity of the inactivated enzyme could be fully recovered in a time-dependent manner by further incubation with NAD+ (but not NADH). It was also found that inhibition by neplanocin A does not involve dissociation of the bound NAD+ or NADH from the enzyme, but simply reduction of the NAD+ to NADH.
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D006867:Hydrolases / Q000037:antagonists & inhibitors*; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence
D009243:NAD; C031096:neplanocin A; D000242:Cyclic AMP; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine