title: Dissection of the biotinyl subunit of transcarboxylase into regions essential for activity and assembly.
abstract: Transcarboxylase, a multisubunit enzyme containing 12 S, 5 S, and 1.3 S subunits, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate (overall reaction) via two partial reactions. In the first partial reaction, a carboxyl group from methylmalonyl-CoA bound to the 12 S subunit is transferred to the biotin of the 1.3 S subunit, and, in the second partial reaction, the carboxylated biotin transfers its carboxyl group from biotin to pyruvate, bound to the 5 S subunit. Previously we have shown that the region around the biotinyl lysine of the 1.3 S subunit is critical for catalysis, that peptides in the amino-terminal region of 1.3 S are capable of forming complexes with 12 S and 5 S, and that amino acids in the carboxyl terminus of the 1.3 S subunit form part of the recognition site for holocarboxylase synthetase. In order to further examine the role of the sequences in this subunit, we generated 8 shortened forms of the 1.3 S biotinyl subunits missing either one or both termini. Truncated 1.3 S subunits were active in both partial reactions until deletion reached amino acid 59. None of the truncated subunits was able to support stable complex formation with the 12 S and 5 S subunits or catalyze the overall reaction. The results suggest that the region between 59 and 78 is required for activity and the sequence 1-18 is required for enzyme assembly. Activity in the partial reactions correlated with intrinsic fluorescence enhancement of tryptophan residues in either the 12 S or 5 S subunit. Fluorescence enhancement was observed with the shortened 1.3 S subunits until truncation reached amino acid 59 implying either 1) that the internal sequence, 59-78, transiently associates with the other subunits to properly orient the biotin for catalysis or 2) that the sequence 59-78 contributes to the folded conformation of the 1.3 S subunit so that subunit interactions can take place.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935.
authors.forename: B C
authors.initials: BC
authors.lastname: Shenoy
authors.forename: G K
authors.initials: GK
authors.lastname: Kumar
authors.forename: D
authors.initials: D
authors.lastname: Samols
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D019878:Carboxyl and Carbamoyl Transferases; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology; D017353:Gene Deletion; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011424:Propionibacterium / Q000201:enzymology; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013050:Spectrometry, Fluorescence; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D011773:Pyruvates; D001710:Biotin; D019289:Pyruvic Acid; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase