A concerted, allosteric model is developed, and equations are derived for quantitative interpretation of the kinetic and equilibrium binding data of rabbit muscle pyruvate kinase at pH 7.5 and 23 degrees C. The simplest model which seems likely to rationalize the experimental data involves two conformational states. In this model, two simplifying assumptions are made. First, the affinities of pyruvate kinase for both substrate and inhibitor are assumed to depend only upon the conformational state of the tetrameric enzyme. Second, the rate of product formation is also assumed to depend only upon the enzyme conformation. All types of experimental data are analyzed simultaneously to estimate the parameters which best predict the total body of experimental results. The fitted parameters indicate that the intrinsic allosteric equilibrium favors the active (R) state by 11 to 1. The substrate phosphoenolpyruvate binds preferentially, by a factor of 10, to the R state, whereas the inhibitor Phe has about 23 times higher affinity for the inactive (T) state. In all cases tested, the calculated values are in good agreement with the experimental data.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.