The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypyruvate, glyoxylate and oxamate, though not carboxylated, still induce the movement. The allosteric activator acetyl-CoA exerts only a slight stimulation on the rate of translocation to the second sub-site, and this stimulation arises from an increase in the dissociation constant for Mg2+.

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The 2',3'-dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg-oATP2-) was shown to be a linear competitive inhibitor with respect to MgATPZin both the acetyl-CoA-dependent and -independent activities of the enzyme but was a non-competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg-oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme. Although bicarbonate, pyruvate and oxaloacetate were ineffective, both MgATP2-and acetyl-CoA protected the enzyme against this chemical modification. At 100% inactivation, 1.1 0.1 mol of Mg-oATPZ-were bound to the enzyme per mole of biotin. Acetyl-CoA had no effect on this stoichiometry.Chromatography of samples of an enzymic digest of Mg-~['~C]ATP~--labelled enzyme revealed one major band of radioactivity which co-chromatographed with authentic Lys-oATP.

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.

The oxidation level of oxalate is such that incorporation of its carbon into cell constituents must involve a net reduction. If it is the sole carbon source for microbial growth then it must also undergo condensation reactions resulting in the synthesis of, for example, C3 and C4 units necessary for formation of carbohydrate and amino acid carbon skeletons (for a review of this aspect, see Kornberg, 1959). Previous work, mainly with whole cells of P8eudomona8 oxalaticus (Quayle & Keech, 1959a, 1960) indicated that a reductive step precedes any condensation reactions, and that this initial reduction reaction is that of oxalylcoenzyme A to glyoxylate. It was also suggested that successive steps involve condensation of two molecules of the glyoxylate to tartronic acid semialdehyde, which in turn is reduced to glyceric acid by a reaction sequence similar to that proposed for the growth of Pseudomona8 species on glycollate (Kornberg & Gotto, 1959). The formation and metabolism of oxalylcoenzyme A, including its reduction to glyoxylate by reduced triphosphopyridine nucleotide in cellfree extracts of P. oxalaticus, are reported in this paper. Part of this work has been presented to the Biochemical Society (Quayle, Keech & Taylor, 1960). METHODS AND MATERIA:LS Maintenance and growth of the organism. The growth conditions have been described by Quayle & Keech (1960). Isotopic materials. Radioactive chemicals were purchased from The Radiochemical Centre, Amersham, Bucks.