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 microM) 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.
Potter
D
D
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Wojnar
J M
JM
Narasimhan
C
C
Miziorko
H M
HM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003497:Cyclic N-Oxides / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D010449:Peptide Mapping; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D051381:Rats; D013113:Spin Labels; D013873:Thionucleotides / Q000378:metabolism
D003497:Cyclic N-Oxides; D018076:DNA, Complementary; D013113:Spin Labels; D013873:Thionucleotides; C096873:adenosine 5'-O-(S-acetamidoproxyl 3-thiotriphosphate); C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D008239:Lysine