Pyruvate carboxylase from Aspergillus nidulans

Abstract

1. 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 M , determined by electrophoresis in the presence of sodium dodecyl sulphate is 133 000 * 5000.2. Electron microscopic analysis of purified A . niduluns 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.3. Significantly better preservation of molecular structure is obtained if A . niduluns 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.4. I -Aspartate, but not 2-oxoglutarate, enhances the rate of inactivation observed on incubation of A. niduluns pyruvate carboxylase at 4 "C in the presence of 0.5 M KC1. 5.Addition of L-aspartate in low concentrations enhances the effectiveness of inhibition by 2-oxoglutarate by causing a decrease in the value of [1]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.6. 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. The data presented also provide further evidence supporting the postulate that I -aspartate and 2-oxoglutarate occupy distinct regulatory sites on A . nidulans pyruvate carboxylase.Previous studies on pyruvate carboxylase purified from Aspergillus niduluns have established that the enzyme which catalyses the reaction Pyruvate + MgATP' -+ HCO; -MgZ

Metabolic Consequences of Altered PhosphoenolpyruvateCarboxykinase Activity in Corynebacterium glutamicum Reveal Anaplerotic Regulation Mechanisms in Vivo

“…Pronk et al (1996) determined the inhibition constant of Saccharomyces cerevisiae pyruvate carboxylase for oxaloacetate as 220 mM. The K i of the Aspergillus nidulans enzyme for a-ketoglutarate is 6.0 mM (Osmani et al, 1984). Modak and Kelly (1985) found 60% inhibition of Rhodobacter capsulatus pyruvate carboxylase by 5 mM a-ketoglutarate, which corresponds to a K i of 3-4 mM.…”

Inhibitors of Pyruvate Carboxylase~!2009-12-01~!2010-02-01~!2010-04-15~!

“…The suggestion of independent binding sites is supported by the fact that the loss of activation by acetyl CoA upon incubation of PC with trinitrobenzene sulfonate does not correlate with any concomitant loss of inhibition by aspartate [93, 98, 99]. PC from the fungus Aspergillus nidulans has also been shown to be partially inhibited by aspartate [98, 100]]. Aspartate is a competitive inhibitor with respect to acetyl CoA and is non-competitive with respect to MgATP and pyruvate [98], consistent with a binding site located in the allosteric domain of the enzyme.…”

“…PC from A. nidulans has been shown to be inhibited by the binding of α-ketoglutarate at a distinct allosteric site removed from the site of action for L-aspartate. [91, 98, 100]. Similarly, PC from avian liver is also inhibited by α-ketoglutarate binding at a distinct inhibitory site from that of glutamate [90].…”

Structure, function and regulation of pyruvate carboxylase

“…Using avidin as a structural probe, Johannssen et al [155] have shown that the biotin moieties are localized in the midline cleft on the external surface of each subunit, close to the inter-subunit junction (see Figure 3b). PCs from Aspergillus nidulans [156], S. cere isiae [157] and even Pseudomonas citronellolis [158], in which PC is arranged as an (αβ) % configuration, all appeared to be tetrahedron-like structures with a midline cleft similar to that in the vertebrate enzymes. Dilution of sheep [77,159] and chicken [160] PCs resulted in inactivation of PC activity, accompanied by dissociation of the active tetramers into inactive dimers and monomers, as revealed by electron microscopic and high-resolution gel-filtration studies.…”