Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding.
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).
Gomi
T
T
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Takata
Y
Y
Date
T
T
Fujioka
M
M
Aksamit
R R
RR
Backlund
P S
PS
Cantoni
G L
GL
D043383:Adenosylhomocysteinase; D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D001051:Apoenzymes; D005971:Glutamates; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D009243:NAD; D001224:Aspartic Acid; D018698:Glutamic Acid; D006867:Hydrolases; D043383:Adenosylhomocysteinase