Functional Evaluation of Conserved Basic Residues in Human Phosphomevalonate Kinase

Abstract

Phosphomevalonate kinase (PMK) catalyzes the cation dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins 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 basicity can result in catalytically impaired enzymes. Based on this precedent, conserved basic residues of human PMK have been mutated and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished V max values in both reaction directions (>1000-fold) with only slight K m perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated K m values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60 and 30-fold, respectively) as well as decreases (50-fold (forward) and 85-fold (reverse)) in V max . R84M also exhibits inflated K m values (50 and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The K i values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45-and 63-fold; effects are comparable to the 30-and 38-fold inflations in K m for mevalonate 5-diphosphate. R141M exhibits little perturbation in V max (14-fold (forward) and 10-fold (reverse)) but has inflated K m values for ATP and ADP (48 and 136-fold, respectively). The K d of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.Phosphomevalonate kinase (PMK 1 ; EC 2.7.4.2) catalyzes the reversible ATP dependent phosphorylation of mevalonate 5-phosphate to produce mevalonate 5-diphosphate and ADP.The reaction represents a key step in the mevalonic acid mediated biosynthesis of isopentenyl diphosphate and other polyisoprenoid metabolites. Disruption of the PMK encoding gene in yeast (1) has demonstrated the essentiality of this enzyme. Enzyme activity was documented in pig liver (2) and initial characterization work employed the purified porcine enzyme (3,4). The sequence of human PMK has been deduced (5) and expression of a recombinant GST- † This work was supported in part by NIDDK

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

“…These biophysical observations contrast with expectations for an ordered sequential mechanism, based of steady state kinetic studies on pig PMK [73]. The possibility of random substrate/product binding to human PMK does, however, agree with the observation of product inhibition of human PMK by mevalonate 5-diphosphate that is competitive with respect to substrate mevalonate 5-phosphate [79]. The preparation of a series of 15 N labeled human PMK proteins containing single arginine substitutions (e.g.…”

“…Work in our lab with the recombinant human protein indicated that Lys-22, as well as Arg-18, have a substantial influence on catalysis in accordance with their location in a P-loop [78]. Evaluation of the importance of other conserved basic residues [79] implicated Arg-110 as making a large contribution to catalysis, Arg-111 and Arg-84 as influencing mevalonate 5-phosphate binding, and Arg-141 as affecting ATP binding. Using the recombinant Streptococcus pneumoniae enzyme, mutations in a series of proposed solvent accessible residues have been characterized [80] and interpreted in the context of a structural model of protein with MgATP and phosphomevalonate ligands.…”

Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase

“…[13–15] Herdendorf and Miziorko investigated the functional role of conserved basic residues in human PMK and suggested that R110 was important for human PMK catalysis. [16] R111 and R84 are situated close to the “Walker B” motif and seem to be involved in binding M5P. [16] Residues K100 and K101 are in close proximity to the ATP binding site, and likely stabilize the transition state for conversion of ATP to ADP.…”

“…[16] R111 and R84 are situated close to the “Walker B” motif and seem to be involved in binding M5P. [16] Residues K100 and K101 are in close proximity to the ATP binding site, and likely stabilize the transition state for conversion of ATP to ADP. [17, 18]…”

NMR Dynamics Investigation of Ligand-Induced Changes of Main and Side-Chain Arginine N−H’s in Human Phosphomevalonate Kinase

“…To facilitate this difficult task, PMK contains 17 arginines (Figure 1) and 8 lysines, with many in the active site to help neutralize the negative charge on the phosphates. The most important arginines, based on site-directed mutagenesis studies, are R18, R48, R73, R84, R110, R111, R141 7, 8. Here we describe the use of NMR dynamics methods to characterize changes to the mobility of arginine sidechains upon ligand binding.…”