An improved purification procedure, an alternative assay and activation of mevalonate kinase by ATP.
An improved procedure for the purification of pig liver mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) is described. A high-voltage electrophoresis assay was developed for mevalonate kinase. The procedure separates mevalonate from phosphomevalonate and also from diphosphomevalonate so that it can be used to measure the subsequent enzyme, phosphomevalonate kinase (EC 2.7.4.2). The assay has allowed the reassessment of the metal ion and nucleotide specificity of the pig liver enzyme. Some of the previously reported properties reflected those of the enzymes in the coupling assay rather than mevalonate kinase itself. A series of compounds were tested as activators or inhibitors of mevalonate kinase. It was found that ATP4-, arsenate and, to a smaller extent, inorganic phosphate activated the enzyme. At fixed MgATP2- (1 mM) concentrations the activation of mevalonate kinase by free ATP4- at pH 8.0 was observed at concentrations at up to 10-fold that of MgATP2- before causing any inhibition. The presence of free ATP4- resulted in a biphasic Lineweaver-Burke plot with apparent Km values for MgATP2- being 0.14 mM and 60 microM, respectively. Fluorescence measurements were consistent with the notion that the binding of excess ATP4- to the enzyme caused a conformational change.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002413:Cations, Divalent; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D002413:Cations, Divalent; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase