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

Negatively stained enzyme‐avidin complexes, seen at different stages of the titration of the biotin‐binding sites on avidin with chicken liver pyruvate carboxylase, have been studied using electron microscopy. Formation of linear, unbranched polymers of the enzyme‐avidin complex is seen to occur 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 prserved. From the structure of the polymers and the observation of single enzyme‐avidin complexes, we deduce that the biotin groups on the enzyme are located on the external faces of each subunit close to, and probably within 3 nm of, the intersubunit junction.