Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme.

A method is described for purification of pyruvate carboxylase from Aspergillus nidulans by affinity chromatography on monomeric avidin-Sepharose. The purified enzyme is homogeneous as judged by electrophoretic and immunochemical analysis. The sub-unit Mr determined by electrophoresis in the presence of sodium dodecyl sulphate is 133000 +/- 5000. Electron microscopic analysis of purified A. nidulans pyruvate carboxylase after negative staining with uranyl acetate reveals the presence of molecules showing rhomboid and triangular projections as well as a projection showing two intensity maxima. A cleft running along the longitudinal axis of the sub-unit is observed in the rhomboid and triangular projections. Interconversion between all three projections can be obtained in a tilt series. Significantly better preservation of molecular structure is obtained if A. nidulans pyruvate carboxylase is prepared for electron microscopy in the presence of acetyl-CoA, 2-oxoglutarate or as the enzyme-avidin complex. L-Aspartate has no significant effect when added alone but markedly decreases the enhanced preservation given by acetyl-CoA. No marked alterations in molecular dimensions are caused by any of these additions. L-Aspartate, but not 2-oxoglutarate, enhances the rate of inactivation observed on incubation of A. nidulans pyruvate carboxylase at 4 degrees C in the presence of 0.5 M KCl. Addition of L-aspartate in low concentrations enhances the effectiveness of inhibition by 2-oxoglutarate by causing a decrease in the value of [I]0.5 without affecting the Hill coefficient h or the extent of activity observed at saturating 2-oxoglutarate concentrations. Conversely addition of low concentrations of 2-oxoglutarate decreases the concentration of L-aspartate required to give 50% inhibition but also causes a fall in h and an absolute increase in the extent of activity observed in the presence of saturating L-aspartate concentrations. The data are consistent with the proposal that A. nidulans pyruvate carboxylase is a tetrameric molecule in which the four sub-units are located at the corners of a tetrahedron. Metabolites which regulate the activity of the enzyme do not cause major alterations in this molecular structure but may alter its stability.(ABSTRACT TRUNCATED AT 400 WORDS)