J. Biol. Chem., Vol. 265, Issue 27, 16102-16107, 09, 1990

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

T Gomi, Y Takata, T Date, M Fujioka, RR Aksamit, PS Backlund Jr and GL Cantoni
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.

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).
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				H. Ando, A. Mizutani, T. Matsu-ura, and K. Mikoshiba IRBIT, a Novel Inositol 1,4,5-Trisphosphate (IP3) Receptor-binding Protein, Is Released from the IP3 Receptor upon IP3 Binding to the Receptor J. Biol. Chem., March 14, 2003; 278(12): 10602 - 10612. [Abstract] [Full Text] [PDF]

				K. E. Bethin, N. Petrovic, and M. J. Ettinger Identification of a Major Hepatic Copper Binding Protein as S-Adenosylhomocysteine Hydrolase J. Biol. Chem., September 1, 1995; 270(35): 20698 - 20702. [Abstract] [Full Text] [PDF]

				C.-S. Yuan and R. T. Borchardt Photoaffinity Labeling of Human Placental S-Adenosylhomocysteine Hydrolase with [2-[IMAGE]H]8-Azido-adenosine J. Biol. Chem., July 7, 1995; 270(27): 16140 - 16146. [Abstract] [Full Text] [PDF]

				J. Komoto, Y. Huang, T. Gomi, H. Ogawa, Y. Takata, M. Fujioka, and F. Takusagawa Effects of Site-directed Mutagenesis on Structure and Function of Recombinant Rat Liver S-Adenosylhomocysteine Hydrolase. CRYSTAL STRUCTURE OF D244E MUTANT ENZYME J. Biol. Chem., October 6, 2000; 275(41): 32147 - 32156. [Abstract] [Full Text] [PDF]