Mevalonic acid is a key intermediate in the biosynthesis of cellular isoprenoids and sterols. Three enzymes, mevalonate kinase, phosphomevalonate kinase, and mevalonate 5-pyrophosphate decarboxylase, are required to metabolize mevalonate to the biosynthetically active isoprenoid, isopentenyl pyrophosphate. Initial work outlined for the enzymes of mevalonate metabolism focuses on mevalonate kinase, which can modulate production of isoprenoids and sterols. Perturbation of mevalonate kinase activity from its normal physiological range has been correlated with disease. In particular, mevalonic aciduria results from inherited mevalonate kinase deficiency. The proposed studies will result in the elucidation of structure/function correlations that account for mevalonate kinase catalysis and feedback regulation. This information will be useful for prediction of the consequences of mutations in the human gene that encodes this enzyme. Additionally, such data will facilitate the design of therapeutic strategies aimed at attenuating enzyme activity and potentially diminishing prenylation of cellular targets. Progress toward these long-term objectives will be generated by initial pursuit of four specific aims. (1) Recombinant forms of human and rat mevalonate kinase will be developed and characterized. The purified enzymes will be kinetically and structurally characterized and the mechanism of feedback inhibition established. (2) Using affinity labeling and sited-directed mutagenesis techniques, mevalonate kinase's active site will be identified and the function of active site amino acids assigned. (3) High resolution structures of rat and human mevalonate kinases will be determined by X-ray crystallography. (4) Human mevalonate kinase mutants will be modeled to determine the molecular basis for mevalonic aciduria.
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