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 co li, The homogeneous enzyme had a specific activity of 30 units/mg and an observed subunit molecular mass of 42 kDa. The Michaelis constants (K-m) for DL-potassium mevalonate (288 mu m) and for ATP (1.24 mM) wer e in agreement with values reported for enzymes isolated from rat live r (Tanaka, R. D., Schafer, B. L., Lee, L. Y., Freudenberger, J. S., an d Mosley, S. T. (1990) J. Biol. Chem, 265, 2391-2398), Recombinant rat mevalonate kinase was inactivated by the lysine-specific reagent, pyr idoxal phosphate (PLP), ATP (5 mM) afforded protection against inactiv ation, suggesting reaction of PLP with an active-site lysine, Mapping, isolation, and Edman degradation of the ATP-protectable peptide from [H-3]PLP-inactivated borohydride-reduced mevalonate kinase allow assig nment of lysine 13, a residue invariant in known mevalonate kinase seq uences, as the modification site, These results represent the first id entification of an active-site residue in mevalonate kinase. The funct ion of lysine 13 was evaluated by replacing this residue with methioni ne, V-m of the mutant protein is diminished by 56-fold, suggesting tha t lysine 13 facilitates catalysis, K-d values of wild-type and mutant proteins for ATP were determined in electron spin resonance competitio n experiments, The observed 56-fold diminution in affinity for the mut ant enzyme supports an additional role for lysine 13 in stabilization of ATP binding.