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.

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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.The Ras GTP-binding proteins play a pivotal role in a variety of signal transduction and differentiation processes (1, 2). Ras is also involved in the generation of a number of human cancers, and several oncogenic point mutations of Ras are known (3, 4). Ras is activated by the conversion of the GDP-bound inactive form to the GTP-bound active form in response to various extracellular signals (5). A variety of extracellular signals can activate mitogen-activated protein kinase (MAP kinase) 1 (also known as extracellular signal-regulated kinase (ERK)) through both Ras-dependent and Ras-independent mechanisms (6). A Ras-dependent pathway linking the epidermal growth factor receptor to MAP kinase, through the protein kinase Raf, has been elucidated (7,8). A Ras-dependent, Raf-independent MAP kinase activation system has also been identified (9, 10).Ras proteins are part of the group of proteins that are post-translationally prenylated (11). In the case of Ras, this modification involves the attachment of the farnesyl group to the protein through a thioether linkage to a cysteine located four residues from the carboxyl terminus, followed by removal of the three carboxyl-terminal amino acids and methylation of the newly exposed ␣-carboxyl group of the farnesyl cysteine residue (12). Additionally, H-Ras and N-Ras, but not K-Ras, undergo palmitoylation at one or more upstream cysteine residues (13). Although necessary for the normal and oncogenic functions of many proteins, including Ras (14), the specific properties imparted by these post-translational modifications have, to a large extent, remained unclear. Prenylation of proteins has been implicated in membrane binding (13, 15) and in protein-protein recognition (16, 17). Furthermore, the relative contribution of each of the processing steps is unknown.Previously we developed a cell-free assay system, derived from Xenopus laevis eggs, to identify a direct target molecule for Ras. In this system, Ras promotes the activation of MAP kinase through MAP kinase kinase/ERK kinase (MEK). Using this system, we have iden...

Carvone and perillaldehyde were shown to inhibit the transformation of Candida albicans to a filamentous form at concentrations far lower and more biologically relevant than the concentrations necessary to inhibit growth. This morphological transformation is associated with C. albicans pathogenicity; hence these naturally occurring monoterpenes are potential lead compounds in the development of therapeutic agents against C. albicans infection.

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