Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding

• Published in: The Journal of biological chemistry 1990-09, Vol.265 (27), p.16102-16107
• Main Authors: Gomi, T, Takata, Y, Date, T, Fujioka, M, Aksamit, R R, Backlund, P S, Cantoni, G L
• Format: Article
• Language: English
• Subjects: Adenosylhomocysteinase; Apoenzymes - isolation & purification; Apoenzymes - metabolism; Aspartic Acid; Base Sequence; Binding Sites; Cloning, Molecular; Escherichia coli - genetics; Exact sciences and technology; Glutamates; Glutamic Acid; Hydrolases - genetics; Hydrolases - isolation & purification; Hydrolases - metabolism; Kinetics; Liver - enzymology; Mathematical analysis; Mathematics; Molecular Sequence Data; Molecular Weight; Mutation; NAD - metabolism; Oligonucleotide Probes; Potential theory; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sciences and techniques of general use
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)46194-1

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme. In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit. The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+. From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme. In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme. Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively. These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site. This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 719-723).