EFFECT OF DELETION FROM THE CARBOXYL-TERMINUS OF THE 12 S-SUBUNIT ON ACTIVITY OF TRANSCARBOXYLASE

Transcarboxylase from Propionibacterium shermanii is a biotin-containi ng 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 CO OH-terminal truncations of the 12 S subunit. Five of these subunits, w hich remained soluble following expression in Escherichia coli and wer e missing from 39 to 97 COOH-terminal amino acids, were purified and c ompared to the full-length subunit after enzyme complexes were assembl ed in vitro. All of the truncated subunits were 90% as active in the t ranscarboxylase reaction as wild type except the reaction containing t he shortest complex, TC-12 S (1-507), which had 54% of the wild type a ctivity (TC-12 S-WT). The reduced activity was not due to a lack of Co A ester binding sites or the K(m) for substrate. However, TC-12 S (1-5 07) was slower to form than TC-12 S-WT and had more incomplete complex es as judged by high performance liquid chromatrography 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 he at-labile compared to wild type. We conclude that the COOH-terminal re gion 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 c omplexes. Once complexes do form, the catalytic capacity of TC-12 S (1 -507) is almost the same as TC-12 S-WT.