David W. Potter

Identification of Catalytic Residues in Human Mevalonate Kinase

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 aV m = 37 units/mg,K m (ATP) = 74 μm, andK m (DL-MVA) = 24 μm. 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 ∼2-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 elevatedK m values for ATP (∼20-fold) and mevalonate (∼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 K d values that are comparable to estimates for wild-type enzyme. The >40,000-fold diminution in k cat 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.

Identification and Functional Characterization of an Active-site Lysine in Mevalonate Kinase

We report the construction of an expression plasmid for rat mevalonate kinase and the overexpression of recombinant enzyme in Escherichia coli. The homogeneous enzyme had a specific activity of 30 units/mg and an observed subunit molecular mass of 42 kDa. The Michaelis constants (Km) for DL-potassium mevalonate (288 μM) and for ATP (1.24 mM) were in agreement with values reported for enzymes isolated from rat liver (Tanaka, R. D., Schafer, B. L., Lee, L. Y., Freudenberger, J. S., and Mosley, S. T. (1990) J. Biol. Chem. 265, 2391-2398). Recombinant rat mevalonate kinase was inactivated by the lysine-specific reagent, pyridoxal phosphate (PLP). ATP (5 mM) afforded protection against inactivation, suggesting reaction of PLP with an active-site lysine. Mapping, isolation, and Edman degradation of the ATP-protectable peptide from [3H]PLP-inactivated borohydride-reduced mevalonate kinase allow assignment of lysine 13, a residue invariant in known mevalonate kinase sequences, as the modification site. These results represent the first identification of an active-site residue in mevalonate kinase. The function of lysine 13 was evaluated by replacing this residue with methionine. Vm of the mutant protein is diminished by 56-fold, suggesting that lysine 13 facilitates catalysis. Kd values of wild-type and mutant proteins for ATP were determined in electron spin resonance competition experiments. The observed 56-fold diminution in affinity for the mutant enzyme supports an additional role for lysine 13 in stabilization of ATP binding.

The influence of heterodimer partner ultraspiracle/retinoid X receptor on the function of ecdysone receptor

A pair of nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP), heterodimerize and transduce ecdysteroid signals. The EcR and its nonsteroidal ligands are being developed for regulation of transgene expression in humans, animals and plants. In mammalian cells, EcR:USP heterodimers can function in the absence of ligand, but EcR/retinoid X receptor (EcR:RXR) heterodimers require the presence of ligand for activation. The heterodimer partner of EcR can influence ligand sensitivity of EcR so that the EcR/Locusta migratoria RXR (EcR:LmRXR) heterodimers are activated at lower concentrations of ligand when compared with the concentrations of ligand required for the activation of EcR/Homo sapiens RXR (EcR:HsRXR) heterodimers. Analysis of chimeric RXRs containing regions of LmRXR and HsRXR and point mutants of HsRXR showed that the amino acid residues present in helix 9 and in the two loops on either end of helix 9 are responsible for improved activity of LmRXR. The EcR:Lm‐HsRXR chimera heterodimer induced reporter genes with nanomolar concentration of ligand compared with the micromolar concentration of ligand required for activating the EcR:HsRXR heterodimer. The EcR:Lm‐HsRXR chimera heterodimer, but not the EcR:HsRXR heterodimer, supported ligand‐dependent induction of reporter gene in a C57BL/6 mouse model.