Substrate and inhibitor specificity of mevalonate kinase determined with substrate analogues

Abstract

Substrate analogues of mevalonate kinase were prepared and their kinetic properties determined with the enzyme from pig liver. The analogues are derived from mevalonate by substitutions of hydroxyl and/or methyl at C3(group 1), by systematic prolongation of C6 to yield 3-alkyl-3,5-dihydroxyvalerates (group 2) and by methylation at C2, C6 and C4 (group 3). Compounds of group 1 were inhibitors of mevalonate kinase, their Ki values ranging over 140 mM to 2.5 mM and decreasing in the series CH2 > C(CH3)2 > CH(OH) > CH(CH3) > C(OH)-CO2H > C(OH)CH2OH. Three of these analogues, the competitive inhibitors 3,5-dihydroxyvalerate, 3-methyl-5-hydroxy-valerate and 3,3-dimethyl-5-hydroxyvalerate are alos (poor) substrates of the kinase. Substances of group 2 contain an additional hydrophobic group and are noncompetitive inhibitors with Ki values decreasing systematically from 7.7 mM to 0.7 mM in the sequence 3-n-propyl, 3-n-butyl, 3-n-pentyl and 3-n-hexyl derivative of 3,5-dihydroxyvalerate. The methylated mevalonates of group 3 are competitive inhibitors as well as good substrates of the kinase, their Ki = Km values ranging over 4.3-0.2 mM and their maximal velocities related to mevalonate varying between 7-65%. All of these analogues have in common the carbon chain and 5-hydroxy group of the substrate and could, therefore, fit to the active site of mevalonate kinase and be phosphorylated by ATP. The results, however, indicate a strong dependence of such interaction on the ligands at C3 of the substrate. Enzymic activity as well as affinity are lost if both of these ligands are replaced by hydrogen; partial restoration occurs at a low level if only one of the ligands is concerned. In the presence of the ligands even monomethylation at C2 and C4 yields enzymically active products. Oxidation of C6 of the substrate yields inhibitors but methylation yields a reasonably good substrate. Thus 3,5-dihydroxy-3-ethylvalerate fits into the active site whereas higher C6 homologues are only inhibitory and probably bind to a hydrophobic region of the enzyme. Some of these results are shown to have parallels in the enzymic behaviour of fluorinated mevalonates.