Purification of a mammalian protein geranylgeranyltransferase. Formation and catalytic properties of an enzyme-geranylgeranyl pyrophosphate complex.
A protein geranylgeranyltransferase (PGGT) that transfers the geranylgeranyl group from geranylgeranyl pyrophosphate (GGPP) to the cysteine residue in the C-terminal sequence Cys-Ali-Ali-Leu (Ali is an aliphatic amino acid) of proteins and peptides has been purified to apparent homogeneity from bovine brain. This was accomplished by affinity chromatography of partially purified enzyme on a gel containing a covalently attached hexapeptide SSCILL. This peptide was identified as a tight-binding ligand of the PGGT by employing a semi-random peptide synthetic strategy. The purified enzyme consists of two subunits of apparent molecular mass 40 and 48 kDa. Affinity-purified PGGT effectively catalyzes the prenylation of peptides that contain a C-terminal Leu or Phe residue. The PGGT forms a stable binary complex with intact GGPP that can be isolated by gel filtration. Addition of a peptide substrate to this complex results in the quantitative transfer of the prenyl group to the peptide. This transfer occurs without the equilibration of enzyme-bound GGPP with free GGPP. When the PGGT was incubated with farnesyl pyrophosphate, the amount of binary complex formed was about 25% of that formed with GGPP.
Yokoyama
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Department of Chemistry, University of Washington, Seattle 98195.
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D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D013379:Substrate Specificity; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism
D011106:Polyisoprenyl Phosphates; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C002963:geranylgeranyl pyrophosphate