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 V m ؍ 37 units/mg, K m(ATP) ؍ 74 M, and K m(DL-MVA) ؍ 24 M. The sensitivity of enzyme to watersoluble 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 ϳ2-fold changes in V m and K m 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 V m and elevated K m values for ATP (ϳ20-fold) and mevalonate (ϳ40-fold). Such effects would be compatible with a role for Biosynthesis of isoprenoids and sterols requires the ATP-dependent phosphorylation of mevalonic acid. In humans, diminished activity of mevalonate kinase (EC 2.7.1.36), the enzyme that catalyzes this reaction (1), results in mevalonic aciduria (2). The enzyme has long been considered to be a cytosolic protein, but recent work on mevalonate kinase has implicated it in peroxisomal metabolism (3). In this context, depressed mevalonate kinase activity has also been correlated with peroxisomal deficiency disorders (4, 5). Although these observations suggest that detailed information on this enzyme would be useful, mevalonate kinase has not received as much attention as other enzymes in the isoprenoid/sterol biosynthetic pathway.Enzymological characterization of mammalian mevalonate kinase has included kinetic work (6), which suggests that the enzyme catalyzes an ordered sequential reaction with mevalonic acid assigned as the first substrate bound and phosphomevalonate as the first product released. Inhibition of the enzyme by geranyl pyrophosphate (7) and farnesyl pyrophosphate (8), metabolites that are formed downstream in the isoprenoid biosynthetic pathway, has been reported, and such inhibition has been suggested to have physiological relevance (9, 10). Although an amino acid substitution that presumably accounts for human mevalonic aciduria has been documented (11), little is known about the active site amino acids that are important to enzyme function. Group-specific reagents have been employed to demonstrate that mevalonate kinase contains reactive cysteine (6) and lysine (12) residues. Recently, the first identification of an active site amino acid was accomplished when protein chemistry and mutagenesis work indicated that lysine-13 influences ATP binding (13).Availability of a recombinant form of human mevalonate kinase would facilitate studies on inherited mutations in this enzyme. Such an enzyme, available in a stable, highly purified form and in substantial amounts, could also be useful for investigation of the structure/function correlations that account for phosphomevalonate production or for feedbac...