RESUMO
Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4A resolution). hMDD exhibits V(max)=6.1+/-0.5 U/mg; K(m) for ATP is 0.69+/-0.07 mM and K(m) for (R,S) mevalonate diphosphate is 28.9+/-3.3 microM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a approximately 1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K(i)=2.3+/-0.3 uM) and 6-fluoromevalonate 5-diphosphate (K(i)=62+/-5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V(max)=0.25+/-0.0 2U/mg and a 15-fold inflation in K(m) for mevalonate diphosphate. N17A's K(i) values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8A) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.
Assuntos
Carboxiliases/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Carboxiliases/genética , Carboxiliases/metabolismo , Domínio Catalítico/genética , Cristalografia por Raios X , Primers do DNA/genética , Humanos , Técnicas In Vitro , Cinética , Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Mevalonate kinase (MK), which catalyzes a key reaction in polyisoprenoid and sterol metabolism in many organisms, is subject to feedback regulation by farnesyl diphosphate and related compounds. The structures of human mevalonate kinase and a binary complex of the rat enzyme incubated with farnesyl thiodiphosphate (FSPP) are reported. Significant FSPP hydrolysis occurs under crystallization conditions; this results in detection of farnesyl thiophosphate (FSP) in the structure of the binary complex. Farnesyl thiodiphosphate competes with substrate ATP to produce feedback inhibition of mevalonate kinase. The binding sites for these metabolites overlap, with the phosphate of FSP nearly superimposed on ATP's beta-phosphate and FSP's polyisoprenoid chain overlapping ATP's adenosine moiety. Several hydrophobic amino acid side chains are positioned near the polyisoprenoid chain of FSP and their functional significance has been evaluated in mutagenesis experiments with human MK, which exhibits the highest reported sensitivity to feedback inhibition. Results suggest that single and double mutations at T104 and I196 produce a significant inflation of the K(i) for FSPP (approximately 40-fold for T104A/I196A). Such an effect persists when K(i) values are normalized for effects on the K(m) for ATP, suggesting that it may be possible to engineer MK proteins with altered sensitivity to feedback inhibition. Comparison of animal MK protein alignments and structures with those of a MK protein from Streptococcus pneumoniae indicates that sequence differences between N- and C-terminal domains correlate with differences in interdomain angles. Bacterial MK proteins exhibit more solvent exposure of feedback inhibitor binding sites and, consequently, weaker binding of these inhibitors.