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Title: Studies of ATP-dependent enzymes in mevalonate pathway
Other Titles: Jia qiang wu suan tu jing zhong ATP yi lai mei de yan jiu
甲羥戊酸途徑中 ATP 依賴酶的研究
Authors: Qiu, Yongge (邱永革)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2006
Publisher: City University of Hong Kong
Subjects: Enzymes
Mevalonic acid
Notes: CityU Call Number: QP601.Q25 2006
Includes bibliographical references (leaves 210-232)
Thesis (Ph.D.)--City University of Hong Kong, 2006
xxii, 232 leaves : ill. (some col.) ; 30 cm.
Type: Thesis
Abstract: A large number of isoprenoids exist naturally and universally in organisms. Many of them are biologically active and participate in diverse cellular process, such as cholesterol, bile acids, ubiquinone, heme A, and dolichol. Isoprenoids are biosynthesized by consecutive condensation of their five-carbon precursor, isopentenyl diphosphate to its isomer, dimethylally diphosphate. Two biosynthetic pathways are known for these precursors: the mevalonate pathway and MEP (2-C-methyl-D-erythritol 4-phosphate) pathway. The mevalonate pathway is essential for animals, fungi, the cytoplasm of phototropic organisms, some eubacteria and Archaea. The mevalonate pathway has been the target for chemotherapy of cardiovascular diseases in human, and a promising target for development of new anticancer and antimicrobial agents. Inhibition of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway, has been fully documented and lead to the application of statins. In this thesis, we report our study of the three consecutive ATP-dependent enzymes in the mevalonate pathway: mevalonate kinase (MVK), phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MDD). These enzymes are all involved, together with HMG-CoA reductase, in regulation of cholesterol biosynthesis in cells, which may be potential targets for inhibition of mevalonate pathway and control of downstream product biosynthesis. The genes of rat phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase were cloned from rat liver cDNA library and constructed into a bacterial expression vector pLM1 with six continuous histidines attached to the end of the genes, which were then overexpressed in E. coli respectively. From the cell lysate, the soluble proteins were purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity. Based on SDS-PAGE analysis, the molecular weights of the proteins were determined to be 22 kDa and 45 kDa. The kinetic properties of the proteins were characterized, and site-directed mutagenesis was used for study of amino acid residues. A 400-fold decrease in activity was found for PMK mutant C92S, suggesting that the residue Cys92 plays an important role in the catalysis. Some highly conserved residues in MDD were also mutated and the resulting proteins were characterized. No detected activity was found with the spectrophotometric assay for mutants K23A, R162A, K208A, D306A/N/E, indicating that these residues all function crucially in the enzymatic reaction. A series of mevalonate analogues were synthesized and characterized. Their interactions with mevalonate kinase were studied, among which some substrates and inhibitors were found. 2-Fluoromevalonate, 6-fluoromevalonate, 2,6-difluoromevalonate, 2,2-difluoromevalonate and 2,4-dihydroxy-2-methyl-butane-1-sulfonate were characterized as substrates of rat and M. jannaschii MVK. Meanwhile, 2,4-dihydroxy-2-methyl-butane-1-sulfonate, replacing carboxyl group in the substrate mevalonate with sulfonate, is a moderate substrate for rat and M. jannaschii MVK, indicating that the role of carboxyl group can be partially replaced by other anions. Analogues with removal of carboxyl group from mevalonate, 3-methyl butane (pentane, hexane)-1,3-diol, are neither substrates nor inhibitors for MVK, suggesting that carboxylate is crucial for substrate binding. These results strongly suggest that an anion (as carboxylate or sulfonate), which will be attracted by a positive-charged center in the active site of the enzyme, is probably indispensable for substrate (or competitive inhibitor) binding. Analogues with removal of 5-hydroxy from mevalonate, 3-methyl-3-hydroxy-pentanate, 3,4-dimethyl-3-hydroxy-pentanate, 5-fluoro-3-methyl-3-hydroxy-pentanate, 3-methyl-3-hydroxy-butanate, 3-methyl-3-hydroxy-3-pentenate, 3-hydroxy-3-phenyl-butyric acid are mild to good competitive inhibitors for rat MVK, suggesting that 5-hydroxy at mevalonate may have little contribution for the binding of the substrate. A series of mevalonate 5-diphosphate analogues (or derivatives) were synthesized and characterized. Their interactions with the three enzymes were also studied. These diphosphate analogues show poor inhibition for MVK. Some of them showed mild to good inhibition on PMK. 2-Fluoromevalonate 5-diphosphate was found to be an irreversible inhibitor on MDD. Its time and concentration-dependent inactivation was characterized with KI (3.02 µM) and kinact (0.72 min-1); and the partition ratio was determined to be 12. The mechanism of the inactivation was proposed as following: the inhibitor is phosphorylated at 3-OH first and then a nucleophilic attack takes place at C-2 by an amino acid residue in the active site of the enzyme to form a covalent-bound complex. Two groups of compounds with combination of mevalonate (analogues or derivatives) and geranyl pyrophosphate were synthesized and characterized. A one-pot procedure was developed for the first time for preparation of this type of compounds. Some of these bifunctional compounds show good inhibition for MVK and MDD at the same time. P’-geranyl 2-fluoromevalonate diphosphate inactivates MDD in a similar way to 2-fluoromevalonate 5-diphosphate; meanwhile it is a good competitive inhibitor of MVK. This type of molecules provide a novel example of a single inhibitor blocking two sequential steps simultaneously in the mevalonate pathway, and may become a useful lead compound for further development for treating cardiovascular diseases and cancers. In conclusion, this thesis describes organic syntheses of a number of compounds, most of which have not been reported. Some novel procedures were developed for preparation of these molecules. Rat PMK and MDD cloning and mutation studies were carried out. Interaction studies of the synthesized compounds with the three enzymes increased our knowledge on the enzymes and their catalyzed reactions. Two irreversible inhibitors of MDD were found, and a type of bifunctional molecules was proved to be good inhibitors of MVK and MDD simultaneously. For drug safety consideration in treatment of cardiovascular diseases, it is necessary to develop drugs other than statins to target different enzymes along the mevalonate pathway. Our findings improve possibility to realize it, which may provide useful information for drug development by pharmaceutical industry.
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