Post-translational regulation of mevalonate kinase by intermediates of the cholesterol and nonsterol isoprene biosynthetic pathways.
To assess the potential for feedback inhibition by isoprene intermediates in the cholesterol and nonsterol isoprene biosynthetic pathway, we expressed human cDNAs encoding mevalonate kinase (MKase), phosphomevalonate kinase (PMKase), and mevalonate diphosphate decarboxylase (MDDase) as fusion proteins in Escherichia coli DH5alpha, and purified these proteins by affinity chromatography. Several phosphorylated and non-phosphorylated isoprenes were analyzed as inhibitors of the enzymes using a standard spectrophotometric assay. Of the three proteins, only MKase was inhibited through competitive interaction at the ATP-binding site. The intermediates studied (and their relative inhibitory capacity) were: geranylgeranyl-diphosphate (GGPP, C20) > farnesyl-diphosphate (FPP, C15) > geranyl-diphosphate (GPP, C10) > isopentenyl-diphosphate (IPP, C5) > or = 3,3-dimethylallyl-diphosphate (DMAPP, C5) > farnesol (C15) > dolichol-phosphate (DP, C(80-100)). Mevalonate-diphosphate, geraniol, and dolichol were not inhibitors. Our data further define the spectrum of physiologic inhibitors of MKase, and provide the first evidence for feedback inhibition of MKase by a nonsterol isoprene produced by the branched pathway, dolichol-phosphate. These results provide additional evidence that MKase may occupy a central regulatory role in the control of cholesterol and nonsterol isoprene biosynthesis.
Hinson
D D
DD
Institute of Metabolic Disease, Baylor University Medical Center, Dallas, TX 75226, USA.
Chambliss
K L
KL
Toth
M J
MJ
Tanaka
R D
RD
Gibson
K M
KM
D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000096:biosynthesis* / Q000494:pharmacology; D004288:Dolichol Phosphates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008798:Mevalonic Acid / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D012717:Sesquiterpenes; D013729:Terpenes / Q000378:metabolism* / Q000494:pharmacology
D004288:Dolichol Phosphates; D004791:Enzyme Inhibitors; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D013729:Terpenes; C004808:farnesyl pyrophosphate; D002784:Cholesterol; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase; D002262:Carboxy-Lyases; C022503:pyrophosphomevalonate decarboxylase; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid