Escherichia coli diacylglycerol kinase is an evolutionarily optimized membrane enzyme and catalyzes direct phosphoryl transfer.
In this contribution the kinetic mechanism and substrate specificity of Escherichia coli diacylglycerol kinase were examined. Steady state kinetic studies were carried out under mixed micellar conditions using a novel continuous coupled assay system. The kinetic data were consistent with a random equilibrium mechanism, implying that diacylglycerol kinase catalyzes direct phosphoryl transfer from MgATP to diacylglycerol. This was supported by failure to detect an enzyme-phosphate covalent intermediate and by the observation that the bisubstrate analog adenosine 5'-tetraphosphoryl-3-O-(1,2-dihexanoyl)-sn-glycerol inhibits the enzyme (Ki << Km,DAG). While diacylglycerol kinase's kcat/Km is modest compared with the efficiency of many water-soluble enzymes, the enzyme nevertheless appears to be an evolutionarily optimized biocatalyst in the sense that its chemical reaction rate approaches the substrate diffusion-controlled limit. The in vivo rate-limiting step of DAGK's reaction appears to be, in part, the transbilayer diffusion of diacylglycerol from the outer leaflet to the inner leaflet of the cytoplasmic membrane where DAGK's active site is located. DAGK was observed to maintain a high nucleotide substrate specificity, with most of this specificity being expressed in the form of reductions in kcat for ATP analogs.
Badola
P
P
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Sanders
C R
CR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D019852:Diacylglycerol Kinase; D004926:Escherichia coli / Q000201:enzymology*; D019143:Evolution, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D009943:Organophosphorus Compounds; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase