Abstract
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.
Highlights
§ To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226
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
Using cDNA that encodes the human enzyme [11], we have developed a useful system for overexpression and isolation of human mevalonate kinase
Summary
§ To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Oxisomal deficiency disorders [4, 5] 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. 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 be useful for investigation of the structure/function correlations that account for phosphomevalonate production or for feedback regulation by geranyl and farnesyl pyrophosphates. Using cDNA that encodes the human enzyme [11], we have developed a useful system for overexpression and isolation of human mevalonate kinase The utility of this recombinant protein is underscored by the results of mutagenesis and enzymological studies, which implicate amino acids important to catalysis of phosphomevalonate production
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