BIOL 125 |
| Phosphomevalonate kinase (PMK) catalyzes a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. Animal PMK proteins are predicted to belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of this basicity results in catalytically impaired enzymes. Based on this precedent, several conserved basic residues of human PMK (K48, R73, R110, R111, R138, R141) have been mutated to replace charged side chains. The mutant proteins have been purified and kinetically characterized. The R138M mutation did not significantly perturb the Vmax or Km values in either reaction direction. K48M and R73M exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight perturbations in Km values(<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution compared to wild-type PMK. R111M has substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60 and 30-fold, respectively) with a moderate (50-fold (forward) and 85-fold (reverse)) decrease in Vmax. The Kis and Kii values for R111M product inhibition by mevalonate 5-diphosphate are inflated 37-fold and 20-fold, respectively when compared with wtPMK; effects are comparable to the 30-fold inflation in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax (14-fold (forward) and 10-fold (reverse)) but has inflated Km values for ATP and ADP (48 and 136-fold, respectively). The Kd of ATP for R141M, as determined by changes in tryptophan fluorescence, was inflated 27-fold compared to wtPMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 contributes to binding of mevalonate 5-phosphate and R141 to binding of ATP. |
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Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biological Chemistry |