Probing the Catalytic Roles of Arg548 and Gln552 in the Carboxyl Transferase Domain of the \u3cem\u3eRhizobium etli\u3c/em\u3e 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. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of 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. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305.Abdussalam Adina-Zada, Sarawut Jitrapakdee, John C. Wallace and Paul V. Attwoo

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. Kinetic studies demonstrated that L-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. L-aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of L-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of L-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. L-aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, L-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and L-aspartate) are spatially distinct but connected by a network of allosteric interactions.Chaiyos Sirithanakorn, Abdussalam Adina-Zada, John C. Wallace, Sarawut Jitrapakdee, and Paul V. Attwoo

Ligand Recognition by the TPR Domain of the Import Factor Toc64 from <i>Arabidopsis thaliana</i>

<div><p>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 <u>T</u>etratrico<u>p</u>eptide <u>R</u>epeat (TPR) domain of the chloroplast import receptor Toc64 from <i>Arabidopsis thaliana</i> (<i>At</i>). The monomeric TPR domain binds with 1∶1 stoichiometry in similar micromolar affinity to both Hsp70 and Hsp90 as determined by isothermal titration calorimetry (ITC). Mutations of the terminal EEVD motif caused a profound decrease in affinity. Additionally, this study considered the contributions of residues upstream as alanine scanning experiments of these residues showed reduced binding affinity. Molecular dynamics simulations of the TPR domain helices upon peptide binding predicted that two helices within the TPR domain move backwards, exposing the cradle surface for interaction with the peptide. Our findings from ITC and molecular dynamics studies suggest that <i>At</i>Toc64_TPR does not discriminate between C-termini peptides of Hsp70 and Hsp90.</p></div

Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation

Abstract not available.Kamonman Choosangtong, Chaiyos Sirithanakorn, Abdul Adina-Zada, John C. Wallace, Sarawut Jitrapakdee, Paul V. Attwoo

Key intrapeptide interactions.

<p><b>A.</b> Intrapeptide interaction in the T_C70 system (coloured green) <b>B.</b> Intrapeptide interaction in the T_C90 system (coloured magenta). The TPR domain is shown as a ribbon diagram in grey.</p

Biophysical characterization of <i>At</i>Toc64_TPR-H6 using size exclusion chromatography and circular dichroism.

<p><b>A.</b> Elution profile of <i>At</i>Toc64_TPR-H6 from size exclusion chromatography using a Superdex 75 prep grade column. The different shaped stars represent different forms of the protein as described in the figure. The Ve/Vo versus LogMW plot for superdex standards is shown as an inset. Numbers 1–4 represent different standard proteins used as described in the methods. Red triangle represents <i>At</i>Toc64_TPR-H6. <b>B.</b> Schematic representation of secondary structure of <i>At</i>Toc64_TPR. <b>C.</b> CD spectrum profile of the protein obtained after analysis with DICHROWEB.</p

Interactions occurring in the protein-peptide interface generated by Ligplot.

<p><b>A.</b> Interactions between the TPR domain and the C-terminal octapeptide of Hsp70; <b>B.</b> Interactions between the TPR domain and C-terminal octapeptide of Hsp90. The peptide is shown in purple bonds and the protein in brown bonds.</p

Analysis of the molecular dynamics trajectory obtained after 50 A.

<p>Root mean square deviation (RMSD) plot for the apo TPR receptor (black trace), the Hsp70 C-terminal octapeptide bound form of the receptor (T_70: green trace) and the Hsp90 C-terminal octapeptide bound form of the receptor (T_C90: magenta trace). <b>B.</b> The atomic positional fluctuation (RMSF) plot obtained for each of the above systems. The loop with high B factors in T_C70 is shown with a green arrow.</p