farnesyl-pyrophosphate and phosphomevalonic-acid

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.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Asteraceae; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Ions; Kinetics; Magnesium; Manganese; Mevalonic Acid; Phosphotransferases (Alcohol Group Acceptor); Polyisoprenyl Phosphates; Sesquiterpenes

cDNA encoding human mevalonate kinase has been overexpressed and the recombinant enzyme isolated. This stable enzyme is a dimer of 42-kDa subunits and exhibits a Vm = 37 units/mg, Km(ATP) = 74 microM, and Km(DL-MVA) = 24 microM. The sensitivity of enzyme to water-soluble carbodiimide modification of carboxyl groups prompted evaluation of four invariant acidic amino acids (Glu-19, Glu-193, Asp-204, and Glu-296) by site-directed mutagenesis. Elimination of Glu-19's carboxyl group (E19A, E19Q) destabilizes the enzyme, whereas E19D is stable but exhibits only approximately 2-fold changes in Vm and Km values. E296Q is a stable enzyme, which exhibits kinetic parameters comparable to those measured for wild-type enzyme. E193A is a labile protein, whereas E193Q is stable, exhibiting >50-fold diminution in Vm and elevated Km values for ATP (approximately 20-fold) and mevalonate (approximately 40-fold). Such effects would be compatible with a role for Glu-193 in interacting with the cation of the MgATP substrate. D204A and D204N are stable enzymes lacking substantial mevalonate kinase activity. The active sites of these Asp-204 mutants are intact, based on their ability to bind a spin-labeled ATP analog with stoichiometries and equilibrium binding constants that are comparable to those determined for wild-type enzyme. Competitive displacement experiments demonstrate that the Asp-204 mutants can bind ATP with Kd values that are comparable to estimates for wild-type enzyme. The >40,000-fold diminution in kcat for the Asp-204 mutants and the demonstration that they contain an otherwise intact active site support assignment of a crucial catalytic role to Asp-204. The assignment of Asp-204 as the catalytic base that facilitates deprotonation of the C-5 hydroxyl of mevalonic acid would be compatible with the experimental observations.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Catalysis; Humans; Kinetics; Mevalonic Acid; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Phosphotransferases (Alcohol Group Acceptor); Polyisoprenyl Phosphates; Protein Conformation; Rats; Recombinant Proteins; Sesquiterpenes; Structure-Activity Relationship

Products of the isoprenoid metabolism were identified upon incubations of extracts from Plasmodium falciparum infected red blood cells with [14C] mevalonate. Uninfected erythrocytes and wild type yeast Saccharomyces cerevisiae extracts were used as controls. In parasitized red blood cells as well as in yeast extracts, mevalonate was converted into the biosynthetic isoprenoid precursors of sterol pathway until farnesyl pyrophosphate. In contrast, no mevalonate conversion was observed in uninfected erythrocyte extracts. The isoprenoid metabolism appeared stage-dependent as shown by the increase of radiolabelled farnesyl pyrophosphate amount at the beginning of the schizogonic phase (30-36 hours).

Topics: Acetates; Animals; Cells, Cultured; Erythrocytes; Hemiterpenes; Humans; Malaria; Mevalonic Acid; Organophosphorus Compounds; Plasmodium falciparum; Polyisoprenyl Phosphates; Saccharomyces cerevisiae; Sesquiterpenes