HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dircks, L. K. Articles by Sul, H. S. Search for Related Content PubMed PubMed Citation Articles by Dircks, L. K. Articles by Sul, H. S. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 49, 34728-34734, December 3, 1999

A Conserved Seven Amino Acid Stretch Important for Murine Mitochondrial Glycerol-3-phosphate Acyltransferase Activity
SIGNIFICANCE OF ARGININE 318 IN CATALYSIS

From the Department of Nutritional Sciences, University of California, Berkeley, California 94720

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. We previously cloned the cDNA sequence to murine mitochondrial GPAT (Yet, S-F., Lee, S., Hahm, Y. T., and Sul, H.S. (1993) Biochemistry 32, 9486-9491). We expressed the protein in insect cells which was targeted to mitochondria, purified, and reconstituted mitochondrial GPAT activity using phospholipids (Yet, S.-F., Moon, Y., and Sul, H. S. (1995) Biochemistry 34, 7303-7310). Deletion of the seven amino acids from mitochondrial GPAT, ³¹²IFLEGTR³¹⁸, which is highly conserved among acyltransferases in glycerolipid biosynthesis, drastically reduced mitochondrial GPAT activity. Treatment of mitochondrial GPAT with arginine-modifying agents, phenylglyoxal and cyclohexanedione, inactivated the enzyme. Two highly conserved arginine residues, Arg-318, in the seven amino stretch, and Arg-278, were identified. Substitution of Arg-318 with either alanine, histidine, or lysine reduced the mitochondrial GPAT activity by over 90%. On the other hand, although substitution of Arg-278 with alanine and histidine decreased mitochondrial GPAT activity by 90%, replacement with lysine reduced activity by only 25%. A substitution of the nonconserved Arg-279 with either alanine, histidine, or lysine did not alter mitochondrial GPAT activity. Moreover, R278K mitochondrial GPAT still showed sensitivity to arginine-modifying agents, as in the case of wild-type mitochondrial GPAT. These results suggest that Arg-318 may be critical for mitochondrial GPAT activity, whereas Arg-278 can be replaced by a basic amino acid. Examination of the other conserved residues in the seven amino acid stretch revealed that Phe-313 and Glu-315 are also important, but conservative substitutions can partially maintain activity; substitution with alanine reduced activity by 83 and 72%, respectively, whereas substituting Phe-313 with tyrosine and Glu-315 with glutamine had even lesser effect. In addition, there was no change in fatty acyl-CoA selectivity. Kinetic analysis of the R318K and R318A mitochondrial GPAT showed an 89 and 95%, respectively, decrease in catalytic efficiency but no major change in substrate binding as indicated by the K_m values for palmitoyl-CoA and glycerol 3-phosphate. These studies indicate importance of the conserved seven amino acid stretch for mitochondrial GPAT activity and the significance of Arg-318 for catalysis.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				R. E. Gimeno and J. Cao Thematic Review Series: Glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity J. Lipid Res., October 1, 2008; 49(10): 2079 - 2088. [Abstract] [Full Text] [PDF]

				T. Harayama, H. Shindou, R. Ogasawara, A. Suwabe, and T. Shimizu Identification of a Novel Noninflammatory Biosynthetic Pathway of Platelet-activating Factor J. Biol. Chem., April 25, 2008; 283(17): 11097 - 11106. [Abstract] [Full Text] [PDF]

				A. P. Beigneux, L. Vergnes, X. Qiao, S. Quatela, R. Davis, S. M. Watkins, R. A. Coleman, R. L. Walzem, M. Philips, K. Reue, et al. Agpat6--a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium J. Lipid Res., April 1, 2006; 47(4): 734 - 744. [Abstract] [Full Text] [PDF]

				D. Hollenback, L. Bonham, L. Law, E. Rossnagle, L. Romero, H. Carew, C. K. Tompkins, D. W. Leung, J. W. Singer, and T. White Substrate specificity of lysophosphatidic acid acyltransferase {beta}--evidence from membrane and whole cell assays J. Lipid Res., March 1, 2006; 47(3): 593 - 604. [Abstract] [Full Text] [PDF]

				D. Linden, L. William-Olsson, M. Rhedin, A.-K. Asztely, J. C. Clapham, and S. Schreyer Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis J. Lipid Res., July 1, 2004; 45(7): 1279 - 1288. [Abstract] [Full Text] [PDF]

				L. E. Hammond, P. A. Gallagher, S. Wang, S. Hiller, K. D. Kluckman, E. L. Posey-Marcos, N. Maeda, and R. A. Coleman Mitochondrial Glycerol-3-Phosphate Acyltransferase-Deficient Mice Have Reduced Weight and Liver Triacylglycerol Content and Altered Glycerolipid Fatty Acid Composition Mol. Cell. Biol., December 1, 2002; 22(23): 8204 - 8214. [Abstract] [Full Text] [PDF]

				V. Zaremberg and C. R. McMaster Differential Partitioning of Lipids Metabolized by Separate Yeast Glycerol-3-phosphate Acyltransferases Reveals That Phospholipase D Generation of Phosphatidic Acid Mediates Sensitivity to Choline-containing Lysolipids and Drugs J. Biol. Chem., October 4, 2002; 277(41): 39035 - 39044. [Abstract] [Full Text] [PDF]

				M. R. Gonzalez-Baro, D. A. Granger, and R. A. Coleman Mitochondrial Glycerol Phosphate Acyltransferase Contains Two Transmembrane Domains with the Active Site in the N-terminal Domain Facing the Cytosol J. Biol. Chem., November 9, 2001; 276(46): 43182 - 43188. [Abstract] [Full Text] [PDF]