A conserved seven amino acid stretch important for murine mitochondrial glycerol-3-phosphate acyltransferase activity. Significance of arginine 318 in catalysis

• Published in: The Journal of biological chemistry 1999-12, Vol.274 (49), p.34728-34734
• Main Authors: Dircks, L K, Ke, J, Sul, H S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - metabolism; Animals; Base Sequence; Blotting, Northern; Blotting, Western; Catalysis; Conserved Sequence; COS Cells; cyclohexanedione; Cyclohexanones - pharmacology; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; glycerol-3-phosphate acyltransferase; Glycerol-3-Phosphate O-Acyltransferase - antagonists & inhibitors; Glycerol-3-Phosphate O-Acyltransferase - chemistry; Glycerol-3-Phosphate O-Acyltransferase - genetics; Glycerol-3-Phosphate O-Acyltransferase - metabolism; glycerolipids; Kinetics; Mice; Mitochondria - enzymology; Molecular Sequence Data; phenylglyoxal; Phenylglyoxal - pharmacology; Plasmids - metabolism; Sequence Homology, Amino Acid; Substrate Specificity; Transfection; Trypsin - pharmacology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.49.34728

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, (312)IFLEGTR(318), 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.