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. 2007 Oct 23;46(42):11780-8.
doi: 10.1021/bi701408t. Epub 2007 Sep 29.

Functional evaluation of conserved basic residues in human phosphomevalonate kinase

Timothy J Herdendorf  1 , Henry M Miziorko
Affiliations

Functional evaluation of conserved basic residues in human phosphomevalonate kinase

Timothy J Herdendorf et al. Biochemistry. .

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. On the basis of 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 Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km 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 Vmax. R84M also exhibits inflated Km values (50- and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki 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 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, 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.

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Figures

Figure 1
Figure 1. Phosphomevalonate kinase sequences
(A) The amino acid sequence of H. sapiens (human) phosphomevalonate kinase is shown and the residues numbered to correspond to the text. Conserved basic residues mutated in this study are shown in bold, blue letters. Conserved basic residues previously mutated and evaluated (–19, 22) are shown in bold, red letters (13). (B) Sequence alignment of animal/invertebrate phosphomevalonate kinases. Conserved basic residues mutated in this study are shown in bold, blue letters. Conserved basic residues previously mutated and evaluated (–19, 22) are shown in bold, red letters (13). All sequences were obtained from public databases. Alignment was generated using the BioEdit program (23). Sequences correspond to the following organisms and accession numbers: H. sapiens (human), Q15126; B. taurus (bovine), Q2KIU2; M. musculus (mouse), Q9D1G2; R. norvegicus (rat), NP_001008353; P. troglodytes (chimpanzee), XP_513842; C. familiaris (dog), XP_855082; M. mulatta (rhesus monkey), XP_001114509; D. rerio (zebra fish), XM_680509.1; X. laevis (African clawed frog), NP_001089752; C. elegans (flatworm), CAD66220; B. mori (domestic silkworm), BAF62110; D. melanogaster (fruit fly), AAF53833; A. gambiae (African malaria mosquito), XP_310779.
Figure 2
Figure 2. SDS-PAGE of wild-type and mutant human PMKs
Lane 1: molecular mass markers (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa. Lanes 2 – 12 contain 10 μg of human PMK proteins corresponding to wt, K48M, K69M, R73M, R84M, R93M, R110M, R111M, R130M, R138M, R141M, respectively.
Figure 3
Figure 3. Scatchard analysis of TNP-ATP binding to wild-type and mutant human PMKs
Panel (A) displays the Scatchard plot for wild-type PMK. Panel (B) displays the Scatchard plot for R110M. Panel (C) displays the Scatchard plot for R141M. Binding measurements were performed under the conditions described in the Methods and in Table 3.
Figure 4
Figure 4. Product inhibition of wild-type and mutant human PMKs
Double-reciprocal plots of the initial velocity for PMK formation of ADP as a function of mevalonate 5-phosphate concentration, measured at different levels of the product inhibitor mevalonate 5-diphosphate. Data were fit to a competitive inhibition model using SigmaPlot 10.0/Enzyme Kinetics 1.3. (Systat Software, Inc.). Panel (A) displays the product inhibition for wild-type PMK. Mevalonate 5-diphosphate concentrations utilized were (●) 0.0 mM, (○) 0.07 mM, (▼) 0.11 mM, (▽) 0.15 mM. Panel (B) displays the product inhibition for R84M PMK. Mevalonate 5-diphosphate concentrations utilized were (●) 0.0 mM, (○) 1.27 mM, (▼) 2.44 mM, (▽) 3.50 mM. Panel (C) displays the product inhibition for R111M PMK. Mevalonate 5-diphosphate concentrations utilized were (●) 0.0 mM, (○) 0.73 mM, (▼) 1.46 mM, (▽) 2.19 mM.
Figure 5
Figure 5. Tryptophan fluorescence quenching of WT and R141M PMK by ATP
The average fractional response of 3 WT(■) concentrations (0.20 – 0.33 μM) and 3 R141M (▲) concentrations (0.28 – 0.66 μM) is plotted as a function of ATP concentration. All measurements were done at 25°C in 1.7 mL 100 mM Tris-Cl, 100 mM KCl, 1 mM DTT at pH 7.5. The concentration of MgCl2 in these assays was 20 and 31 mM for WT and R141M, respectively. Samples were excited at 295 nm and the emission was monitored between 310 – 450 nm. For data analysis, values measured at the fluorescent emission peak of ~333 nm were corrected for buffer background fluorescence and for dilution. These data were analyzed by nonlinear regression and fit to a 2-site model [y = Bmax1[X]/(Kd1 + [X]) + Bmax2[X]/(Kd2 + [X])] using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego California USA, (www.graphpad.com). Error bars represent the standard deviation of the three different protein concentrations.
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