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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 Ka pyruvate for the pyruvate-stimulated release of Pi 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 Pi 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 different polypeptide chain within the tetramer.

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. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.