Identification and characterization of a factor which is essential for assembly of transcarboxylase.
Transcarboxylase (TC) from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. It is composed of a central, hexameric 12S subunit with six outer, dimeric 5S subunits held in a stable 26S complex by twelve 1.3S biotinyl subunits. Each of these subunits has been cloned from the P. shermanii genome and expressed in Escherichia coli. The purified, expressed recombinant proteins are all indistinguishable from their authentic counterparts except for the recombinant 5S subunit (termed 5S WT), which does not form TC complexes or catalyze the overall transcarboxylase reaction. Circular dichroism and isoelectric focusing suggested differences existed between the authentic and E. coli-expressed 5S proteins. HPLC gel filtration was used to separate the authentic 5S dimer from additional components in the preparation. 5S dimer thus purified was unable to form TC complexes or catalyze the overall reaction, behaving identically to the recombinant 5S WT subunit. Fractions from the HPLC gel-filtration purification of authentic 5S were then added to 5S WT or 5S dimer, and one fraction was identified which catalyzed the assembly of TC complexes with either 5S preparation. This assembly activity was shown to be dependent on the concentration of this HPLC fraction. Assembly-promoting factor (APF) is heat-stable and probably a protein, on the basis of its protease susceptibility. Studies with APF and the other TC subunits demonstrate its ability to promote complex formation with 12S and 1.3S subunits. Since the APF was purified from crystals of 26S TC, we believe it to be a novel, previously unidentified subunit of transcarboxylase.
Shenoy
B C
BC
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Xie
Y
Y
Sha
D
D
Samols
D
D
D001710:Biotin / Q000032:analysis; D019878:Carboxyl and Carbamoyl Transferases; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D011424:Propionibacterium / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014166:Transferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000648:ultrastructure
D046911:Macromolecular Substances; D001710:Biotin; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase