Kinetic and Thermodynamic Contributions to an Intermolecular Mechanism of Subunit Communication: Coordination of Pyruvate Carboxylase Activity Among Spatially Distinct Active Sites

Catalysis occurring in a multifunctional enzyme at spatially distinct active sites is controlled by an array of factors, including the structure of the enzyme, ligand binding, and productive interaction of substrates to facilitate turnover. Successful execution of the catalytic cycle is partially dependent upon the ability of spatially and functionally discrete active sites to communicate with one another, as well as with any allosteric regulatory regions of the enzyme. This type of long-range communication typically manifests measurable effects on substrate binding or product release. In the case of pyruvate carboxylase (PC), pyruvate binding to the carboxyl transferase (CT) domain induces translocation of the biotin carboxyl carrier (BCCP) domain and subsequently increases the rate of Pi release in the biotin carboxylase (BC) domain. While the kinetic mechanism and structural arrangement of the PC tetramer has largely been elucidated, the source of the intermolecular signals required to facilitate catalysis between distinct active sites remains unclear. The BC and CT domain active sites necessary to produce one oxaloacetate are located on two separate polypeptide chains, while binding of acetyl-CoA in its pocket formed between the allosteric domain and the BC domain is required for stimulation of the overall catalytic rate. In metabolic regulatory enzymes such as PC, it is essential to understand not only the overall mechanism of intersubunit communication, but also the thermodynamic driving forces behind each individual ligand relationship in order to piece together the network of amino acids and subunit domains that is responsible for the dramatic stimulatory response elicited upon binding of acetyl-CoA, the enzyme's essential allosteric activator. Ultimately, this would allow for elucidation of the molecular regulatory mechanism of PC and for subsequent development of therapeutic strategies to target the chronic hyperglycemia associated with its uncontrolled activity in Type 2 diabetics. To address how pyruvate occupancy in the CT domain impacts the behavior of other domains, we generated mixed hybrid tetramers using mutants of the catalytically relevant residues Glu218 (in the BC domain) and Thr882 (in the CT domain) and measured both the pyruvate carboxylation and inorganic phosphate release activities. Our results, which compared the apparent Ka pyruvate for pyruvate-stimulated Pi release catalyzed by the T882S:E218A(1:1) hybrid tetramer to that of the wild-type and the T882S homotetramer, were consistent with an intermolecular mechanism of subunit communication, whereby pyruvate binding at the T882S CT domain was responsible for inducing translocation of the E218A BCCP domain within the same face of the tetramer. We also determined the thermodynamic-linkage of each ligand of PC, that is, the extent to which the presence of one bound substrate or effector positively or negatively influences enzyme turnover in the presence of saturating and subsaturating concentrations of another. The ability of either MgATP or pyruvate to increase the affinity of PC for the other is observed in the presence of acetyl-CoA, while this relationship is entirely lost in its absence. These results have the potential to further reveal the nature of intersubunit communication, in that the enzyme's spatially distinct active sites, even in the presence of the preferred substrates, cannot communicate or coordinate productive catalytic coupling in the absence of the activator. Long-term implications of this proposal include determination of the consequences of imbalanced metabolic flux, such as that observed in Type 2 Diabetes, on the regulatory mechanism and catalytic activity of PC in the liver

Kinetic and Thermodynamic Contributions to an Intermolecular Mechanism of Subunit Communication: Coordination of Pyruvate Carboxylase Activity Among Spatially Distinct Active Sites

Catalysis occurring in a multifunctional enzyme at spatially distinct active sites is controlled by an array of factors, including the structure of the enzyme, ligand binding, and productive interaction of substrates to facilitate turnover. Successful execution of the catalytic cycle is partially dependent upon the ability of spatially and functionally discrete active sites to communicate with one another, as well as with any allosteric regulatory regions of the enzyme. This type of long-range communication typically manifests measurable effects on substrate binding or product release. In the case of pyruvate carboxylase (PC), pyruvate binding to the carboxyl transferase (CT) domain induces translocation of the biotin carboxyl carrier (BCCP) domain and subsequently increases the rate of Pi release in the biotin carboxylase (BC) domain. While the kinetic mechanism and structural arrangement of the PC tetramer has largely been elucidated, the source of the intermolecular signals required to facilitate catalysis between distinct active sites remains unclear. The BC and CT domain active sites necessary to produce one oxaloacetate are located on two separate polypeptide chains, while binding of acetyl-CoA in its pocket formed between the allosteric domain and the BC domain is required for stimulation of the overall catalytic rate. In metabolic regulatory enzymes such as PC, it is essential to understand not only the overall mechanism of intersubunit communication, but also the thermodynamic driving forces behind each individual ligand relationship in order to piece together the network of amino acids and subunit domains that is responsible for the dramatic stimulatory response elicited upon binding of acetyl-CoA, the enzyme's essential allosteric activator. Ultimately, this would allow for elucidation of the molecular regulatory mechanism of PC and for subsequent development of therapeutic strategies to target the chronic hyperglycemia associated with its uncontrolled activity in Type 2 diabetics. To address how pyruvate occupancy in the CT domain impacts the behavior of other domains, we generated mixed hybrid tetramers using mutants of the catalytically relevant residues Glu218 (in the BC domain) and Thr882 (in the CT domain) and measured both the pyruvate carboxylation and inorganic phosphate release activities. Our results, which compared the apparent Ka pyruvate for pyruvate-stimulated Pi release catalyzed by the T882S:E218A(1:1) hybrid tetramer to that of the wild-type and the T882S homotetramer, were consistent with an intermolecular mechanism of subunit communication, whereby pyruvate binding at the T882S CT domain was responsible for inducing translocation of the E218A BCCP domain within the same face of the tetramer. We also determined the thermodynamic-linkage of each ligand of PC, that is, the extent to which the presence of one bound substrate or effector positively or negatively influences enzyme turnover in the presence of saturating and subsaturating concentrations of another. The ability of either MgATP or pyruvate to increase the affinity of PC for the other is observed in the presence of acetyl-CoA, while this relationship is entirely lost in its absence. These results have the potential to further reveal the nature of intersubunit communication, in that the enzyme's spatially distinct active sites, even in the presence of the preferred substrates, cannot communicate or coordinate productive catalytic coupling in the absence of the activator. Long-term implications of this proposal include determination of the consequences of imbalanced metabolic flux, such as that observed in Type 2 Diabetes, on the regulatory mechanism and catalytic activity of PC in the liver

Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism

Protein structure, ligand binding, and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is “sensed” by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent <i>K</i>_a pyruvate for the pyruvate-stimulated release of P_i catalyzed by the T882S:E218A_[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to P_i release for the WT:T882S_[1:1] and E218A:T882S_[1:1] hybrid tetramer-catalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is “sensed” by domains on a <i>different</i> polypeptide chain within the tetramer

Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of <i>Staphylococcus aureus</i> Pyruvate Carboxylase

Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the <i>Staphylococcus aureus</i> PC (<i>Sa</i>PC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the <i>Sa</i>PC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer