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 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.
Potter
D
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Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Miziorko
H M
HM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes; D013329:Structure-Activity Relationship
D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C045038:phosphomevalonic acid; C004808:farnesyl pyrophosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D008798:Mevalonic Acid