Purification and properties of geranylgeranyl-diphosphate synthase from bovine brain

Geranylgeranyl-diphosphate synthase was purified to homogeneity from bovine brain in a one-step procedure employing an affinity column. For the construction of the affinity column, a farnesyl diphosphate analog, O-(6-amino-1-hexyl)-P-farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affi-Gel 10 via the amino group. The native enzyme appeared to be a homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pI for the enzyme was 6.2. The Km values for dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were estimated to be 33, 0.80, and 0.74 microM, respectively. The Km value for isopentenyl diphosphate in the reaction with isopentenyl diphosphate and farnesyl diphosphate was 2 microM. The reaction velocities for the formation of geranylgeranyl diphosphate from dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were in the ratio of 0.004:0.145:1. The intermediate farnesyl diphosphate was formed in the reaction with geranyl diphosphate as an allylic primer. Geranylgeranyl diphosphate acted as a competitive inhibitor against farnesyl diphosphate with an approximate Ki value of 1.2 microM in the condensation reaction of farnesyl diphosphate with isopentenyl diphosphate. Farnesyl-diphosphate synthase catalyzing the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate was also purified to homogeneity from the same organ by similar affinity chromatography using a geranyl diphosphate analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate, as a ligand. This enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that geranylgeranyl diphosphate, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from dimethylallyl diphosphate and isopentenyl diphosphate by the action of farnesyl-diphosphate synthase catalyzing the reaction of C5-->C15, followed by the action of geranylgeranyl-diphosphate synthase catalyzing a single reaction of C15-->C20, and that geranylgeranyl diphosphate can down-regulate its own synthesis through the inhibition of the geranylgeranyldiphosphate synthase action.