Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements.
An enzyme capable of specifically modifying, with a geranylgeranyl isoprenoid, candidate proteins containing a consensus prenylation sequence ending in leucine has been purified from bovine brain. This protein geranylgeranyltransferase (PGGT), isolated using affinity chromatography on an immobilized peptide column, contains two subunits with molecular masses of 48 and 43 kDa, designated alpha and beta, respectively. An antiserum raised to the alpha subunit of the related enzyme, protein farnesyltransferase (PFT), also recognizes this chromatographically identical alpha-subunit of the PGGT by immunoblot analysis. The PGGT and PFT enzymes from bovine brain are shown to be dependent on both Mg2+ and Zn2+ for optimal activity. Demonstration of the Zn2+ dependence of the enzymes requires prolonged incubation or purification in the presence of a chelating agent; we therefore propose that these enzymes be placed into the category of metalloenzymes. Under optimal assay conditions, these enzymes show high specificity toward their prenyl diphosphate substrates, with only a weak competition observed with farnesyl diphosphate in the PGGT reaction or geranylgeranyl diphosphate in the PFT reaction. The two enzymes are differentially sensitive to several detergents tested to determine suitable ones for product stabilization in the reactions. These results confirm previous predictions on the subunit structure of the PGGT and provide an avenue to initiating a molecular analysis of the geranylgeranyl modification of many mammalian proteins.
Moomaw
J F
JF
Section of Cell Growth, Regulation and Oncogenesis, Duke University Medical Center, Durham, North Carolina 27710.
Casey
P J
PJ
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*
D003902:Detergents; D046911:Macromolecular Substances; D004492:Edetic Acid; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C064911:p21(ras) farnesyl-protein transferase; D019204:GTP-Binding Proteins; D008274:Magnesium; D015032:Zinc