Rat liver S-adenosylhomocysteinase. Spectrophotometric study of coenzyme binding.
Rat liver S-adenosylhomocysteinase, a homotetramer, was resolved by treatment with acid ammonium sulfate into apoenzyme and NAD. The apoenzyme thus prepared retained a tetrameric structure but differed in the mobility on nondenaturing polyacrylamide gel electrophoresis. The inactive apoenzyme was reactivated upon incubation with NAD. The restoration of activity paralleled with the tight binding of NAD to apoenzyme, and full activity was obtained when 4 mol of NAD were bound per mol of apoenzyme. The kinetics of reconstitution were apparently biphasic and suggest the existence of two conformers in a slow equilibrium, one of which binds the coenzyme rapidly while the other does so very slowly, if at all. In addition to NAD, apoadenosylhomocysteinase tightly bound nicotinamide hypoxanthine dinucleotide, 3-acetylpyridine adenine dinucleotide and nicotinic acid-adenine dinucleotide. NADP was not bound. Catalytic activity was found only with the enzyme reconstituted with NAD or nicotinamide hypoxanthine dinucleotide. The spectral change observed on interaction of apoadenosylhomocysteinase with NAD was similar to those seen with adenine nucleotides, and was largely approximated by the addition of dioxane to aqueous solutions of adenine nucleotides. By comparison of the difference spectra, it is suggested that the adenine portion of the coenzyme is bound in the hydrophobic pocket of the protein, and that the binding is accompanied by perturbation of tryptophan residue of the protein.
Gomi
T
T
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Takata
Y
Y
Fujioka
M
M
D043383:Adenosylhomocysteinase; D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002942:Circular Dichroism; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D001059:Apoproteins; D009243:NAD; D006867:Hydrolases; D043383:Adenosylhomocysteinase