Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase.
The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca(2+) or Mg(2+) ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 degrees C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent K(m) = 6.5 mol %) and saturation kinetics with respect to CTP (apparent K(m) = 0.3 mm). dCTP was both a substrate (apparent K(m) = 0.4 mm) and competitive inhibitor (apparent K(i) = 0.4 mm) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.
Han
Gil-Soo
GS
Department of Food Science and the Rutgers Center for Lipid Research, Rutgers University, New Brunswick, NJ 08901, USA.
O'Hara
Laura
L
Siniossoglou
Symeon
S
Carman
George M
GM
D000483:Alleles; D002412:Cations / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism*; D019852:Diacylglycerol Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004795:Enzyme Stability; D005559:Formamides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D017830:Octoxynol / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000737:chemistry / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D002412:Cations; D005559:Formamides; D010712:Phosphatidic Acids; D047428:Protein Kinase Inhibitors; C035404:N-ethylformamide; D003570:Cytidine Triphosphate; D017830:Octoxynol; D019852:Diacylglycerol Kinase