Transcarboxylase: XI. ELECTRON MICROSCOPY AND SUBUNIT STRUCTURE

Abstract

Abstract The molecular weights of transcarboxylase and its constituent subunits have been determined by sedimentation equilibrium and by gel electrophoresis and the organization of the subunits has been deduced from electron micrographs. The intact enzyme (molecular weight ∼ 792,000, s20,w ∼ 18 S) presents a profile with a large central subunit to which are attached on one side three smaller peripheral subunits. The isolated central subunit (s20,w ∼ 12 S, molecular weight ∼ 360,000) presents two profiles in the electron microscope, one circular (100 A) with a central hole, the other rectangular (70 x 100 A). It dissociates further at pH 9 to give three subunits (s20,w ∼ 6 S, molecular weight ∼ 120,000) with roughly square profiles (70 x 70 A). In sodium dodecyl sulfate, it gives peptide chains of molecular weight 60,000; therefore, it is proposed that the central subunit is a trimer (symmetry C3) of the 6 S subunits each containing two peptide chains. The three smaller peripheral structures (s20,w ∼ 6 S, molecular weight ∼ 144,000) show 55 x 90 A profiles. Each consists of two metal-containing (cobalt, zinc) peptides of 60,000 molecular weight as well as two carboxyl carrier proteins (s20,w ∼ 1.3 S, molecular weight ∼ 12,000) containing biotin. Thus, transcarboxylase is made up of 18 peptides, 6 metals, and 6 biotins. The three peripheral subunits appear to be attached to the central subunit by an extended peptide chain of about 10 to 15 residues, most likely provided by the carboxyl carrier protein. The flexibility of the structure and the lability of the linkage account for the large variety of profiles observed in the electron microscope. Under mild conditions the enzyme dissociates to a modified enzyme of s20,w ∼ 16 S by loss of one of the peripheral subunits. This 16 S component resembles the silhouette of the head of Mickey Mouse in the electron microscope. The intact transcarboxylase rarely shows 3-fold symmetry but this symmetry is seen in complexes of avidin with transcarboxylase in which the central subunit is surrounded by 3 molecules of avidin alternating with the 3 peripheral subunits. The location of the avidin molecules at the ends of the long axis of the peripheral subunit suggests that the carboxyl carrier proteins are also in these positions and extend to one surface of the central subunit. Reconstitution of the transcarboxylase from the isolated central subunit and the peripheral subunit gives active enzyme with typical profiles of transcarboxylase. However, when the enzyme is dissociated in the absence of glycerol and is then reconstituted at pH 5, a new enzymatically active product (s20,w ∼ 24 S) is formed which has two sets of peripheral subunits on the opposite faces of the central subunit, suggesting that by this treatment, additional binding sites for the peripheral subunit have been exposed on the central subunit.