title: Active site determination of yeast geranylgeranyl protein transferase type I expressed in Escherichia coli.
abstract: The ram2 and cal1 genes encode the alpha and beta subunits of yeast geranylgeranyl protein transferase type I (GGPT-I), respectively. Arginine 166 of the beta subunit was changed to isoleucine (betaR166I), histidine 216 to aspartic acid (betaH216D), and asparagine 282 to alanine (betaN282A) by sequential PCR using mutagenic primers. The mutants were expressed under the same conditions as the wild-type and were assayed for GGPT-I activity. Wild-type yeast GGPT-I, alphaH145D, alphaD140N, betaR166I, betaH216D and betaN282A mutant GGPT-Is were partially purified by ammonium sulfate fractionation followed by a Q-Sepharose column. Characterization studies were performed using the active fraction of the Q-Sepharose column. In the chemical modification reactions, the catalytic activity of purified enzyme decreased in proportion to the concentration of modifying reagents, such as phenylglyoxal and diethyl pyrocarbonate (DEPC). Geranylgeranyl pyrophosphate (GGPP) protected the enzyme activity from the modification with phenylglyoxal. The measurement of GGPP binding to wild-type and five mutant GGPT-Is was performed by a gel-filtration assay. The binding of GGPP to the betaR166I mutant was low and the Km value for GGPP in the betaR166I mutant increased about 29-fold. Therefore, the results suggest a role for this arginine residue that directly influences the GGPP binding. The activity of the DEPC-modified GGPT-I was inhibited by 80% at 5 mM DEPC. The differential absorption at 242 nm may suggest that at this concentration the modified histidine residues were 1.5 mol per GGPT-I. The protein substrate, glutathione S-transferase fused undecapeptide (GST-CAIL) protected the enzyme from inactivation by DEPC, and the Km value for GST-CAIL in the betaH216D mutant increased about 12-fold. The trypsin digestion of [14C]DEPC-modified enzyme yielded a single radioactive peptide. As a result of the sequence of this radioactive peptide, the histidine 216 residue was assumed to be an essential part of binding of peptide substrate.
authors.affiliation: Department of Chemistry, College of Natural Science, Seoul National University, Korea.
authors.forename: H
authors.initials: H
authors.lastname: Kim
authors.forename: C H
authors.initials: CH
authors.lastname: Yang
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002687:Chitin Synthase; D004047:Diethyl Pyrocarbonate; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D006639:Histidine; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010658:Phenylglyoxal; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D017368:Protein Prenylation; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014166:Transferases; D015003:Yeasts / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D011106:Polyisoprenyl Phosphates; C077378:RAM2 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D014166:Transferases; C069390:CHS3 protein, S cerevisiae; D002687:Chitin Synthase; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D004047:Diethyl Pyrocarbonate; C002963:geranylgeranyl pyrophosphate; D010658:Phenylglyoxal