| Autor: |
S B, Woo, B C, Shenoy, H G, Wood, W J, Magner, G K, Kumar, H, Beegen, D, Samols |
| Rok vydání: |
1993 |
| Předmět: |
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| Zdroj: |
The Journal of biological chemistry. 268(22) |
| ISSN: |
0021-9258 |
| Popis: |
Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. The central hexameric 12 S subunit of the enzyme associates with six 6 S subunits in the complete enzyme complex. We have constructed a series of cloned genes which encode COOH-terminal truncations of the 12 S subunit. Five of these subunits, which remained soluble following expression in Escherichia coli and were missing from 39 to 97 COOH-terminal amino acids, were purified and compared to the full-length subunit after enzyme complexes were assembled in vitro. All of the truncated subunits were 90% as active in the transcarboxylase reaction as wild type except the reaction containing the shortest complex, TC-12 S (1-507), which had 54% of the wild type activity (TC-12 S-WT). The reduced activity was not due to a lack of CoA ester binding sites or the Km for substrate. However, TC-12 S (1-507) was slower to form than TC-12 S-WT and had more incomplete complexes as judged by high performance liquid chromatography gel filtration profiles and electron microscopy. Isolated TC-12 S (1-507) was 70-80% as active as TC-12 S-WT. We also noted that the truncated form was heat-labile compared to wild type. We conclude that the COOH-terminal region of the 12 S subunit plays a role in assembly and stability of the hexamer and also affects the binding of 6 S subunits to form enzyme complexes. Once complexes do form, the catalytic capacity of TC-12 S (1-507) is almost the same as TC-12 S-WT. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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