Pharmacological control of the mevalonate pathway: effect on arterial smooth muscle cell proliferation.

The mevalonate (MVA) pathway is involved in cell proliferation. We investigated drugs acting at different enzymatic steps on rat aorta smooth muscle cell (SMC) proliferation. Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (0.1-10 microM) dose-dependently decreased (up to 90%) SMC proliferation. This effect was prevented by 100 microM MVA, 10 microM all-trans farnesol (F-OH) and 5 microM all-trans geranylgeraniol (GG-OH), precursors of protein prenyl groups, but not by 2-cis GG-OH, precursor of dolichols, squalene and ubiquinone. The same inhibitory effect was obtained with 6-fluoromevalonate (1-50 microM), an inhibitor of MVA-pyrophosphate decarboxylase. Partial recovery of cell proliferation was possible by all-trans F-OH and all-trans GG-OH, but not MVA. Squalestatin 1 (1-25 microM), a potent squalene synthase inhibitor, blocked cholesterol synthesis and slightly inhibited (21% decrease) SMC proliferation only at the highest tested concentration. NB-598 (1-10 microM), a potent squalene epoxidase inhibitor, blocked cholesterol synthesis without affecting SMC proliferation. Finally, the benzodiazepine peptidomimetic BZA-5B (10-100 microM), a specific inhibitor of protein farnesyltransferase, time- and dose-dependently decreased SMC proliferation (up to 62%) after 9 days. This effect of BZA-5B was prevented by MVA and all-trans GG-OH, but not by all-trans F-OH. SMC proliferation was not affected by the closely related compound BZA-7B, which does not inhibit protein farnesyltransferase. Altogether, these findings focus the role of the MVA pathway in cell proliferation and call attention to the involvement of specific isoprenoid metabolites, probably through farnesylated and geranylgeranylated proteins, in the control of this cellular event.

The farnesyl group of H-Ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase.

To study the function of the farnesyl modification of Ras, the farnesyl group and a variety of its structural analogs, which lack one or more double bonds and/or the methyl groups, were enzymatically incorporated into recombinant H-Ras in vitro. These proteins were used in a cell- and membrane-free, Ras-dependent mitogen-activated protein kinase (MAP kinase) activation system derived from Xenopus laevis eggs to examine the contribution of the farnesyl group toward the activation of the kinase. Whereas non-farnesylated H-Ras is unable to activate MAP kinase, farnesylation of H-Ras alone, in the absence of further processing, is sufficient to cause the activation of MAP kinase in this system. All of the analogs of the farnesyl group, when incorporated into H-Ras, support the activation of the kinase to variable extents. These results suggest a direct but fairly nonspecific interaction of the farnesyl moiety of H-Ras with a soluble upstream activator of MAP kinase.

Isolation and characterization of an active-site peptide from a monoterpene cyclase labeled with a mechanism-based inhibitor.

(+)-Pinene synthase and (+)-bornyl pyrophosphate synthase from culinary sage (Salvia officinalis L.: Lamiaceae) catalyze the coupled isomerization and cyclization of geranyl pyrophosphate to the indicated bicyclic monoterpenes. The reaction parameters for these monoterpene cyclases have been well defined but the two enzymes have proved difficult to separate and purify in sufficient amounts for detailed structural characterization. A method was developed for the isolation of the two cyclases from highly enriched sage leaf oil gland extracts and for the efficient copurification of the two enzymes to about 95% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); the preparation yielded two overlapping species on two-dimensional polyacrylamide gel electrophoresis as expected. The cyclases were copurified and labeled with the highly selective mechanism-based irreversible inactivator 6-[1-3H]cyclopropylidene-3E-methyl-hex-2-en-1-yl pyrophosphate, subjected to cleavage with CNBr, and the resulting covalently modified peptides were isolated by SDS-PAGE for blotting to a polyvinylidene difluoride membrane and N-terminal amino acid sequence analysis. A radiochemically abundant 5-kDa peptide of the cleavage mixture was shown to be highly homologous, through 22 residues, to a segment (Leu197-Glu218) of (-)-4S-limonene synthase from spearmint (Mentha spicata L.: Lamiaceae), the only monoterpene cyclase for which the complete deduced amino acid sequence is known. These results illustrate the use of the mechanism-based inhibitor for purification and structural studies with the monoterpene cyclases, and they define a presumptive active site region that bears a highly conserved sequence among these enzymes from the mint (Lamiaceae) family.

Mammalian protein geranylgeranyltransferase-I: substrate specificity, kinetic mechanism, metal requirements, and affinity labeling.

Protein geranylgeranyltransferase-I (PGGT-I) catalyzes the transfer of the 20-carbon prenyl group from geranylgeranyl pyrophosphate to the cysteine residue near the C-termini of a variety of eukaryotic proteins. Kinetic analysis of homogenous PGGT-I from bovine brain reveals that the reaction follows a sequential pathway in which either prenyl donor or acceptor can bind first to the enzyme and that the reaction operates at steady-state rather than at rapid equilibrium. Substrate inhibition by prenyl acceptor but not by prenyl donor suggests that geranylgeranyl pyrophosphate binding first to free enzyme is the kinetically preferred pathway. This is supported by isotope trapping experiments which show that the ternary complex goes on to products faster than the release of geranylgeranyl pyrophosphate from the complex. The KM for the interaction of geranylgeranyl pyrophosphate with PGGT-I is markedly affected by the structure of the prenyl acceptor bound to the enzyme. A detailed analysis of the substrate specificity of PGGT-I reveals that peptides which contain a C-terminal leucine are preferred (kcat/KM = 1-5 x 10(5) M-1 s-1) to those that end in serine (kcat/KM = 2-4 x 10(3) M-1 s-1) or phenylalanine (kcat/KM = 0.5 x 10(3) M-1 s-1). PGGT-I also catalyzes the farnesylation of peptides that have a C-terminal leucine; kcat for farnesylation and KM for farnesyl pyrophosphate are similar to those for geranylgeranylation, but the KM for the peptide is 30-fold higher. Geranyl pyrophosphate is utilized by PGGT-I but is a poor substrate. Optimal activity of PGGT-I is obtained in the presence of micromolar amounts of Zn2+ and mM amounts of Mg2+. Mn2+ or Cd2+ but not Co2+ can substitute for Zn2+ and for Mg2+. Metals are not required for tight-binding of geranylgeranyl pyrophosphate to PGGT-I, and the measured dissociation equilibrium constant for this binary complex is 16 nM. Photoaffinity analogues of geranylgeranyl pyrophosphate and farnesyl pyrophosphate were prepared and shown to exclusively label the beta-subunit. The implication of the results for the substrate specificity of protein prenylation in cells is briefly discussed.

Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.