Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies.
The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].
D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010649:Phenylalanine / Q000378:metabolism / Q000494:pharmacology*; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013438:Sulfhydryl Compounds; D013816:Thermodynamics; D014364:Tryptophan; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D010649:Phenylalanine; D010728:Phosphoenolpyruvate; D014364:Tryptophan; D011770:Pyruvate Kinase