Pig liver phosphomevalonate kinase. 1. Purification and properties

Abstract

Pig liver phosphomevalonate kinase (EC 2.7.4.2) has been purified to homogeneity as shown by polyacrylamide gel electrophoresis. The molecular weight estimates range from 21,000 to 22,500. Each molecule is composed of one polypeptide chain. The presence of SH-containing reagents is essential for the preservation of enzymes activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM have been obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, one cysteine residue per molecule of enzyme, and the absence of methionine. The results obtained suggest that the enzyme plays no regulatory function in cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.

Molecular Cloning of Human Phosphomevalonate Kinase and Identification of a Consensus Peroxisomal Targeting Sequence

“…The open reading frame of the human liver cDNA predicts a protein of 192 amino acids with a molecular weight of 21,862 consistent with previous reports for mammalian PMKase (M r ϭ 21,000 -22,500) (12,13). Comparison of the predicted amino acid sequence from the cDNA with that obtained from amino acid sequencing indicates that a single methionine is removed from the N terminus upon maturation of the protein.…”

“…The deduced M r of the protein encoded by the cDNA is 21,862, which is in good agreement with the M r of the purified porcine liver PMKase previously presented (12,13). The cDNA encoding PMKase was fused to a pGEX expression vector system with a bacterial host.…”

“…A cDNA encoding PMKase from yeast has been presented, which encodes a protein with a predicted M r ϭ 48,000. However, the purified PMKase from porcine liver has been reported as a monomeric protein of M r ϭ 22,000 (12,13). This divergence from yeast to mammals seems unusual.…”

Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase.

“…Previous characterizations of the porcine phosphomevalonate kinase had shown it to be 22 kDa (6,32). This difference in protein size was surprising, because to date comparisons between yeast and mammalian clones for other enzymes of sterol biosynthesis, including HMG CoA synthase (27a), HMG CoA reductase (5), mevalonate kinase (51a), farnesyl diphosphate synthetase (3), and lanosterol demethylase (28), have revealed strong conserva- Another feature of phosphomevalonate kinase deduced from the ERG8 nucleotide sequence was the location of a small peptide that may represent a portion of the enzyme active site.…”

“…Isoprene-derived molecules are also used in some species for the covalent modification of tRNAs (17) and specific proteins, including ras and a-factor in S. cerevisiae (1,27,46). Phosphomevalonate kinase from pig liver has been purified to homogeneity and shown to be a monomeric protein with a molecular mass of 22 kDa (6,32). It exhibits strong nucleotide specificity for ATP and utilizes Mg2`as a preferred cofactor.…”