IDENTIFICATION AND CHARACTERIZATION OF A FACTOR WHICH IS ESSENTIAL FOR ASSEMBLY OF TRANSCARBOXYLASE

Transcarboxylase (TC) from Propionibacterium shermanii is a biotin-con taining enzyme which catalyzes the reversible transfer of a carboxyl g roup from methylmalonyl-CoA to pyruvate. It is composed of a central, hexameric 12S subunit with six outer, dimeric 5S subunits held in a st able 26S complex by twelve 1.3S biotinyl subunits. Each of these subun its has been cloned from the P. shermanii genome and expressed in Esch erichia coli. The purified, expressed recombinant proteins are all ind istinguishable from their authentic counterparts except for the recomb inant 5S subunit (termed 5S WT), which does not form TC complexes or c atalyze the overall transcarboxylase reaction. Circular dichroism and isoelectric focusing suggested differences existed between the authent ic and E. coli-expressed 5S proteins. HPLC gel filtration was used to separate the authentic 5S dimer from additional components in the prep aration. 5S dimer thus purified was unable to form TC complexes or cat alyze the overall reaction, behaving identically to the recombinant 5S WT subunit. Fractions from the HPLC gel-filtration purification of au thentic 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) i s heat-stable and probably a protein, on the basis of its protease sus ceptibility. Studies with APF and the other TC subunits demonstrate it s ability to promote complex formation with 12S and 1.3S subunits. Sin ce the APF was purified from crystals of 26S TC, we believe it to be a novel, previously unidentified subunit of transcarboxylase.