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

Abstract

Negatively stained enzyme-avidin complexes, seen at different stages of the titration of the biotin-binding sites on avidin with chicken liver pyruvate carboxylase, were studied using EM. Formation of linear, unbranched polymers of the enzyme-avidin complex occurs when the ratio of avidin to enzyme is between 2:1 and 1:2; beyond these limits only single enzyme tetramers are visible. The single avidin molecules observed seem to have a cuboid structure. The orientation and dimensions of the enzyme tetramers within the polymers indicate that the tetrahedron-like structure, observed in the single molecules, has been preserved. From the structure of the polymers and the observation of single enzyme-avidin complexes, the biotin groups on the enzyme are probably located on the external faces of each subunit close to and probably within 3 nm of the intersubunit junction.