Effect of deletion from the carboxyl terminus of the 12 S subunit on activity of transcarboxylase.
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.
Woo
S B
SB
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Shenoy
B C
BC
Wood
H G
HG
Magner
W J
WJ
Kumar
G K
GK
Beegen
H
H
Samols
D
D
D000214:Acyl Coenzyme A / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019878:Carboxyl and Carbamoyl Transferases; D002264:Carboxylic Acids; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology; D017384:Sequence Deletion; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D000214:Acyl Coenzyme A; D002264:Carboxylic Acids; D004269:DNA, Bacterial; C015357:methylmalonyl-coenzyme A; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase