Further Electron Microscope Studies on Pyruvate Carboxylase

Abstract

Using negative staining and electron microscopic tilting techniques in conjunction with modelling experiments, the fine structure of chicken, sheep and rat pyruvate carboxylases has been studied. The overall configuration appears to be a tetrahedron‐like structure consisting of two pairs of subunits in two different planes orthogonal to each other with the opposing pairs of subunits interacting with each other on their convex surfaces. The predominant form of the enzyme particles mounted and stained in the presence of acetylcoenzyme A consisted of a compact, triangular outline enclosing three readily visible intensity maxima. When samples were mounted in the absence of acetyl‐coenzyme A the molecules were more ‘open’ predominantly rhomboid structures. From tilting experiments it is concluded that the rhomboid images found in the absence of acetyl‐coenzyme A represent partly or wholly flattened forms of the tetrahedron‐like molecule. A feature of the enzyme when mounted in the absence of acetyl‐coenzyme A was the existence of a ‘cleft’ along the longitudinal midline of each subunit, suggesting that the subunits may consist of two distinct domains.

Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes

“…1 a this ratio is 9 : 1, and here only single enzyme-tetramer -avidin complexes can be seen and the enzyme molecules are in the tight tetrahedron-like configuration observed by Mayer et al [4]. These particles have a diameter of 15 -16.5 nm, which agrees with previous measurements [4]. There are numerous free avidin molecules visible and, in general, it is difficult to see those which are bound to the enzyme tetramers because they may be partially obscured by the enzyme.…”

“…Mayer et al [4] were later able to confirm the tetrahedron-like structure and found that acetylCoA stabilised this form of the enzyme. Another feature of this work was the observation of a cleft along the longitudinal midline of each subunit when the enzyme was mounted in the absence of acetyl-CoA.…”

“…Within each pair the binding areas are separated by a distance of about 2.0 nm [6,7] and the pairs are centrally located on the faces of the cuboid. Together with the tetrahedron-like arrangement of the pyruvate carboxylase subunits [4], this makes the binding of Enzyme tetramers are linked together by avidin molecules, with each pair of binding sites on an avidin molecule binding two biotin groups from the adjacent enzyme tetramers. It can be seen that the enzyme tetramers can only exhibit one orientation, with two subunits in contact with the support, when the apparent rectangular profile of the tetramer is evident.…”

“…The reason for this is that the single molecules usually have three subunits in contact with the support [4], see Fig. 6c.…”

“…In the presence of acetyl-CoA where the tetrahedral form of the molecule is observed, the cleft is less visible. Mayer et al [4] suggested that each subunit is divided into two domains with the active site located in the cleft region and that acetyl-CoA held the domains together. There was, however, no information to indicate the location of the active site along the longitudinal axis of the subunit.…”