title: Photoaffinity-labeling peptide substrates for farnesyl-protein transferase and the intersubunit location of the active site.
abstract: --CAAX motif peptides, which are substrates for isoprenylation, were synthetically derivatized with the light-sensitive benzophenone (Bz) group in order to determine their potential use as catalytic site-directed covalent photocross-linking ligands for one of the enzymes catalyzing protein isoprenylation, farnesyl-protein transferase (FPTase). Bz-peptides could be synthesized with [3H]benzophenone and possessed eiter one or two benzophenone groups located at or near the peptide's NH2 terminus (e.g. the mono-Bz probes Bz-ACVIM and Bz-LPCVVM, and the di-Bz derivatized probe Bz-GY-(Bz)PCVVM, referred to as Bz2-GYPCVVM). Each type of derivatized peptide behaved as a substrate for farnesylation in vitro without irradiation, while under 366-nm irradiation each demonstrated covalent cross-linking ability as a catalytic site-directed photoaffinity ligand with tissue-purified or enriched but impure fractions from rat and bovine brain FPTase, as well as with a recombinant human FPTase variant, FPTase (beta alpha t) expressed in Escherichia coli. Without photoactivation, Bz-ACVIM yielded a Kd of 37 nM for the cloned variant of human FPTase. Pseudo first-order photolytic inhibition of FPTase preparations with Bz-peptides, as well as protection from photoinactivation by unmodified -CAAX motif peptides, supported the capacity of these Bz-peptides to serve as co-substrates and their specificity for seeking the catalytic site of the enzyme. SDS-polyacrylamide gel electrophoresis analysis subsequent to photolysis indicated that the mono-Bz-derivatized peptides (e.g. [3H]Bz-LPCVVM or 3H]Bz-ACVIM) became covalently cross-linked preferentially to the approximately 49-kDa beta subunit of the alpha beta dimeric FPTase. The farnesyl-PP cosubstrate bound equally well to unmodified and Bz-ACVIM-labeled enzyme. The di-Bz derivative, [3H]Bz2-GYPCVVM, in contrast, revealed exclusive photocovalent cross-linking with a species of molecular mass approximately 95-97 kDa, indicating that both FPTase subunits were tethered together covalently by the di-Bz probe. Similar differential SDS-polyacrylamide gel electrophoresis cross-linking patterns were obtained with homogeneous FPTases as well as with partially purified rat or bovine brain enzyme preparations. The absence of nonspecific photolabeling of any proteins in the partially purified rat or bovine brain enzyme preparations other than FPTase independently attested to the high efficiency of photocross-linking of the FTPase, and the selective catalytic site-seeking ability of these Bz-derivatized peptide substrates, verifying their potential as structural probes for the active site domain on the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Laboratories of Metabolic Regulation, Boston Biomedical Research Institute, Massachusetts 02114.
authors.forename: W
authors.initials: W
authors.lastname: Ying
authors.forename: L
authors.initials: L
authors.lastname: Sepp-Lorenzino
authors.forename: K
authors.initials: K
authors.lastname: Cai
authors.forename: P
authors.initials: P
authors.lastname: Aloise
authors.forename: P S
authors.initials: PS
authors.lastname: Coleman
mesh_terms: D000345:Affinity Labels; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D017368:Protein Prenylation; D051381:Rats; D013379:Substrate Specificity; D014166:Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D009842:Oligopeptides; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase