Purification and characterization of mevalonate kinase from suspension-cultured cells of Catharanthus roseus (L.) G. Don.
Mevalonate kinase was purified to homogeneity from Catharanthus roseus (L.) G. Don suspension-cultured cells. The purified enzyme had an M(r) of 104,600 and a subunit size of about 41,500. Kinetic studies indicated an ordered sequential mechanism of action, in which mevalonate was the first substrate to bind and ADP was the last product to leave the enzyme. True values for the kinetic constants were determined for mevalonate, with K(ma) = 76 microM and K(ia) = 74 microM, and for ATP, with K(mb) = 0.13 mM and K(ib) = 0. 13 mM; the true V(max) was calculated to be 138.7 nkat/mg of protein. Product inhibition was only detectable at rather high concentrations: above 0.7 mM for 5-phosphomevalonate and above 2 mM for ADP, with an ADP/ATP ratio of at least 1. Mevalonate kinase activity was shown to be strongly inhibited by farnesyl diphosphate. Farnesyl diphosphate acted as a competitive inhibitor toward ATP, with a K(i) value of 0.1 microM. Mevalonate kinase activity was dependent on the presence of divalent ions. At a concentration of 2 mM, Mg(2+) and Mn(2+) were best and equally effective in sustaining activity; compared to Mg(2+) and Mn(2+), relative activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar concentrations of Zn(2+), Fe(2+), Co(2+), Ca(2+), and Ni(2+), respectively. The pH-dependent activity profile of mevalonate kinase showed a broad pH optimum between pH 7 and 10, with a maximum at about pH 8.9.
Schulte
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Division of Pharmacognosy, Gorlaeus Laboratories, Leiden, 2300 RA, The Netherlands. Annelies.Schulte@DeRuiterSeeds.com
van der Heijden
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Verpoorte
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019659:Asteraceae / Q000201:enzymology*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000302:isolation & purification*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012717:Sesquiterpenes
D007477:Ions; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C045038:phosphomevalonic acid; D008345:Manganese; D000244:Adenosine Diphosphate; C004808:farnesyl pyrophosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D008274:Magnesium; D008798:Mevalonic Acid