Phosphomevalonate kinase: functional investigation of the recombinant human enzyme.
Phosphomevalonate kinase (PMK) catalyzes a key step in isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. To expedite functional and structural study of this enzyme, an expression plasmid encoding His-tagged human PMK has been constructed and recombinant enzyme isolated in an active, stable form. PMK catalyzes a reversible reaction; kinetic constants of human PMK have been determined for both forward (formation of mevalonate 5-diphosphate) and reverse (formation of mevalonate 5-phosphate) reactions. Animal and invertebrate PMKs are not orthologous to plant, fungal, or bacterial PMKs, limiting the information available from sequence alignment analysis. A homology model for the structure of human PMK has been generated. The model conforms to a nucleoside monophosphate kinase family fold. This result, together with sequence comparisons of animal and invertebrate PMKs, suggests an N-terminal basic residue rich sequence as a possible "Walker A" ATP binding motif. The functions of four basic (K17, R18, K19, K22) residues and one acidic (D23) residue in the conserved sequence have been tested by mutagenesis and characterization of isolated mutant proteins. Substrate K(m) values for K17M, R18Q, K19M, and D23N have been measured for forward and reverse reactions; in comparison with wild-type PMK values, only modest (<12-fold) changes are observed. In contrast, R18Q exhibits a V(max) decrease of 100/300-fold (forward/reverse reaction). K22M activity is too low for measurement at nonsaturating substrate concentration; specific activity is decreased by >10000-fold in both forward/reverse reactions, suggesting an active site location and an important role in phosphoryl transfer.
Herdendorf
Timothy J
TJ
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
Miziorko
Henry M
HM
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010770:Phosphotransferases / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology
D011994:Recombinant Proteins; D010770:Phosphotransferases; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase