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 Mr 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 Kma = 76 μM and Kia = 74 μM, and for ATP, with Kmb = 0.13 mM and Kib = 0.13 mM; the true Vmax 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 Ki value of 0.1 μM. Mevalonate kinase activity was dependent on the presence of divalent ions. At a concentration of 2 mM, Mg2+ and Mn2+ were best and equally effective in sustaining activity; compared to Mg2+ and Mn2+, relative activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar concentrations of Zn2+, Fe2+, Co2+, Ca2+, and Ni2+, 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.