A conserved seven amino acid stretch important for murine mitochondrial glycerol-3-phosphate acyltransferase activity. Significance of arginine 318 in catalysis.
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.
Dircks
L K
LK
Department of Nutritional Sciences, University of California, Berkeley, California 94720, USA.
Ke
J
J
Sul
H S
HS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D017124:Conserved Sequence; D003512:Cyclohexanones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010658:Phenylglyoxal / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D003512:Cyclohexanones; D004791:Enzyme Inhibitors; D005992:Glycerol-3-Phosphate O-Acyltransferase; D014357:Trypsin; D010658:Phenylglyoxal