D. Potter et al., IDENTIFICATION AND FUNCTIONAL-CHARACTERIZATION OF AN ACTIVE-SITE LYSINE IN MEVALONATE KINASE, The Journal of biological chemistry, 272(9), 1997, pp. 5741-5746
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.