Dissection of the biotinyl subunit of transcarboxylase into regions essential for activity and assembly
| Autor: | B C, Shenoy, G K, Kumar, D, Samols |
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| Rok vydání: | 1993 |
| Předmět: |
Binding Sites
Macromolecular Substances Molecular Sequence Data Propionibacterium Biotin Catalysis Microscopy Electron Spectrometry Fluorescence Mutagenesis Transferases Carboxyl and Carbamoyl Transferases Pyruvic Acid Escherichia coli Amino Acid Sequence Pyruvates Chromatography High Pressure Liquid Conserved Sequence Gene Deletion |
| Zdroj: | The Journal of biological chemistry. 268(3) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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