PURIFICATION AND PROPERTIES OF GERANYLGERANYL-DIPHOSPHATE SYNTHASE FROM BOVINE BRAIN

Geranylgeranyl-diphosphate synthase was purified to homogeneity from b ovine 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 synthes ized and linked to the spacer of the matrix of Affi-Gel 10 via the ami no 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 enz yme was 6.2. The K-m values for dimethylallyl diphosphate, geranyl dip hosphate, and farnesyl diphosphate were estimated to be 33, 0.80, and 0.74 mu M, respectively. The K-m value for isopentenyl diphosphate in the reaction with isopentenyl diphosphate and farnesyl diphosphate was 2 mu M. The reaction velocities for the formation of geranylgeranyl d iphosphate from dimethylallyl diphosphate, geranyl diphosphate, and fa rnesyl diphosphate were in the ratio of 0.004:0.145:1. The intermediat e farnesyl diphosphate was formed in the reaction with geranyl diphosp hate as an allylic primer. Geranylgeranyl diphosphate acted as a compe titive inhibitor against farnesyl diphosphate with an approximate K-i value of 1.2 mu M in the condensation reaction of farnesyl diphosphate with isopentenyl diphosphate. Farnesyl-diphosphate synthase catalyzin g 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 diphosp hate analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate, a s a ligand. This enzyme was a homodimer with a monomeric molecular mas s of 40.0 kDa. These results indicate that geranylgeranyl diphosphate, a lipid precursor for the biosynthesis of a majority of prenylated pr oteins, is synthesized from dimethylallyl diphosphate and isopentenyl diphosphate by the action of farnesyl-diphosphate synthase catalyzing the reaction of C-5 --> C-15, followed by the action of geranylgeranyl diphosphate synthase catalyzing a single reaction of C-15 --> C-20, a nd that geranylgeranyl diphosphate can down-regulate its own synthesis through the inhibition of the geranylgeranyl-diphosphate synthase act ion.