Characterization of the kinetics and activation thermodynamics of intra- and inter-organism hybrid tetramers of pyruvate carboxylase.

In sedimentation velocity experiments, we have been able to detect hybrid Rhizobium etli pyruvate carboxylase tetramers formed between subunits that contain covalently bound biotin and mutant subunits that do not. This was performed by forming complexes of the tetramers with the biotin-binding protein avidin. In addition, we have shown that it is possible to form hybrid tetramers of pyruvate carboxylase subunits from two different organisms (bacteria - Rhizobium etli and fungi - Aspergillus nidulans).

Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate.

L-aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of L-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase.

Coordinating role of His216 in MgATP binding and cleavage in pyruvate carboxylase.

His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3'-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N.

Ligand recognition by the TPR domain of the import factor Toc64 from Arabidopsis thaliana.

The specific targeting of protein to organelles is achieved by targeting signals being recognised by their cognate receptors. Cytosolic chaperones, bound to precursor proteins, are recognized by specific receptors of the import machinery enabling transport into the specific organelle. The aim of this study was to gain greater insight into the mode of recognition of the C-termini of Hsp70 and Hsp90 chaperones by the Tetratricopeptide Repeat (TPR) domain of the chloroplast import receptor Toc64 from Arabidopsis thaliana (At).

Roles of Arg427 and Arg472 in the binding and allosteric effects of acetyl CoA in pyruvate carboxylase.

Mutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation constant (K(a)) of acetyl CoA, with the increase being greater for the Arg472 mutants. These results indicate that while both these residues are involved in the binding of acetyl CoA to the enzyme, Arg472 is more important than Arg427. The mutations had substantially smaller effects on the k(cat) for pyruvate carboxylation.

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA.

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R.

Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA.

In this review we examine the effects of the allosteric activator, acetyl CoA on both the structure and catalytic activities of pyruvate carboxylase. We describe how the binding of acetyl CoA produces gross changes to the quaternary and tertiary structures of the enzyme that are visible in the electron microscope. These changes serve to stabilize the tetrameric structure of the enzyme.

Novel insights into the biotin carboxylase domain reactions of pyruvate carboxylase from Rhizobium etli.

The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353).

Probing the allosteric activation of pyruvate carboxylase using 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA.

2',3'-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme.

Probing the catalytic roles of Arg548 and Gln552 in the carboxyl transferase domain of the Rhizobium etli pyruvate carboxylase by site-directed mutagenesis.

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water.

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase.

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by (1)H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by (31)P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction.

Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme.

Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112.

Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers.

The native form of pyruvate carboxylase is an alpha4 tetramer but the tetramerisation domain of each subunit is currently unknown. To identify this domain we co-expressed yeast pyruvate carboxylase 1 isozyme (Pyc1) with an N-terminal myc tag, together with constructs encoding either the biotin carboxylase (BC) domain or the transcarboxylase-biotin carboxyl carrier domain (TC-BCC), each with an N-terminal 9-histidine tag. From tag-affinity chromatography experiments, the subunit contacts within the tetramer were identified to be primarily located in the 55 kDa BC domain.

Differential regulation of the yeast isozymes of pyruvate carboxylase and the locus of action of acetyl CoA.

Unlike other eukaryotes studied to date, yeast has two genes for pyruvate carboxylase coding for very similar, but not identical, isozymes (Pyc1 and Pyc2), both of which are located in the cytoplasm. We have found that there are marked differences in the kinetic properties of the isozymes potentially leading to differential regulation of Pyc1 and Pyc2 activity by both activators and substrates. For example, Pyc2 is only activated 3.