Abstract
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.
Highlights
The cysteine residue of aCAAX sequence (C, cysteine; A, aliphatic amino acid;X, methionineor serine) at the C-terminal region of precursorproteins
Farnesyl-diphos- widely occurring prenyltransferase in mammalian tissues.On phate synthase catalyzing the formation of farnesyl the other hand, the biosynthesis of geranylgeranyl-PP, whose diphosphatefrom dimethylallydliphosphateand isopen- carbon chain length islonger by one isoprene unit than thaot f tenyl diphosphatewas alsopurified to homogeneityfrom farnesyl-PP, has not been well understood
We have enzyme wasa homodimer with a monomeric molecular massof 40.0kDa. These results indicate that geranylgeranyl diphosphate, a lipid precursor for the biodemonstrated the separation of geranylgeranyl-PP synthase from farnesyl-PP synthase by ion exchange chromatography synthesis of a majority of prenylated proteins, is synthe- followed by hydroxylapatite chromatography of crude extracts sized from dimethylallyl diphosphate and isopentenyl from pig liver [11].Recently, we have reported the separadiphosphate by the action of farnesyl-diphosphatesyn- tionbetween the two enzymes by chromatography of crude c, thase catalyzingthe reaction of -+ CIS,followedby the extracts from rat liver [12] and from bovine brain [13]
Summary
7.0) containing 1mM MgCl,, 10 mM 2-mercaptoethanol, and 0.25 mM sodium azide until use. For purification of geranylgeranyl-PP synthase, the resulting supernatant was brought to 40% saturation with solid ammonium sulfate, stirred for 30 min, and centrifuged at ported t o be effective in purifying farnesyl-PP synthase [18]. When the 4040% ammonium sulfate fraction containing geranyl-transferring and farnesyl-transferring activities was loaded onto the column under the same conditions as reported in a. Another enzyme with farnesyl-transferring activity was recovered with a linear gradienotf farnesyl-PP (0-500p~) These results suggested that geranylgeranyl-PP synthase can bind to the farnesylmethyl affinity gel in thepresence of inorprotocols were carried out at a flow rate of 30 mvh. The fractions having the enzyme activity were combined, concentrated with a Centricon-3 concentrator apparatus, washed with standard buffer three nyl-PP synthase can be released from the gel by farnesyl-PP.
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