The allosteric transitions of rabbit muscle phosphorylase b were analyzed in the temperature range from 29 to 4° by means of kinetic and binding studies. The binding of 5′-adenosine monophosphate to phosphorylase b was studied using a Sephadex gel filtration technique. A computer was programmed to calculate best fits to experimental curves using equations for the two-state model of Monod et al. (Monod, J., Wyman, J., and Changeux, J.-P. (1965), J. Mol. Biol. 12, 88). The results indicated values of L for the ratio T :R of phosphorylase b in the absence of ligands ranging from 6100 at 29° to 11 at 4°. The values for L’ for the ratio T :R in the presence of the substrate anion, glucose 1-phosphate, ranged from 52 at 29° to 0.05 at 4°. At low temperatures phosphorylase dimer b associates. The aggregated (tetrameric) forms of phosphorylase b bind 5′-adenosine monophosphate tighter. Kequil of [tetramer b]/[dimer b]2 was determined by light scattering and by ultracentrifugal sedimentation velocity measurements. The association-dissociation equilibrium was taken into consideration in the theoretical treatment of the data. Aside from substrate anions various other anions and divalent cations function as allosteric effectors and promote the T to R transition of phosphorylase b. The interaction of the glycerol phosphate buffer anion with phosphorylase b was taken into account in the theoretical treatment. Mathematical analysis of kinetic data revealed an activity of phosphorylase b at 1 × 10−5 M 5′-adenosine monophosphate that was too low considering that phosphorylase b is a K system. The evidence suggested less than theoretically expected heterotropic cooperativity of glucose 1-phosphate and 5′-adenosine monophosphate in sodium glycerol phosphate buffer. In order to account for the deviations from theory a scheme is proposed that assumes that phosphorylase b in sodium glycerol phosphate buffer may exist in three states. State T binds little to glucose 1-phosphate and not at all to 5′-adenosine monophosphate. State R as compared with state T binds exclusively to 5′-adenosine monophosphate and preferentially to anionic substrates (i.e., glucose 1-phosphate). State R’ as compared with state R binds more tightly to 5′-adenosine monophosphate but less tightly to glucose 1-phosphate. The existence of two R states of phosphorylase b and a is also deduced from a study of the temperature dependency of the allosteric equilibria and of the Kassn values for 5′-adenosine monophosphate. In the case of phosphorylase b, the transition from R to R′ correlates well with the shift to tetrameric structures (or aggregates of higher order) at high enzyme concentrations (13-18 mg/ml) and at temperatures below 13°. It is suggested therefore that phosphorylase dimer b in state R′ readily associates in the presence of 5′-adenosine monophosphate at low temperatures. This is in contrast to phosphorylase a where the transition from R to R’ occurs around 23° and where the dimeric forms R and the corresponding tetrameric form R (or for that matter, the dimer R’ and the tetramer R’), have similar binding properties for 5’- adenosine monophosphate. Dimer a and tetramer a can each undergo transitions from one form, R, to another form, R’, with temperature. Hence association of dimer a to tetramer a linked to the binding of 5′-adenosine monophosphate does not seem to play a major role, whereas the dimer b → tetramer b association is strongly dependent upon 5′-adenosine monophosphate. The vastly different values for the allosteric T :R equilibrium of phosphorylase b where L is 2100 at 23° and of phosphorylase a where L is 3-13 at this temperature may well explain the equally large differences in the requirements of the unphosphorylated (b) and the phosphorylated (a) enzyme for 5′-adenosine monophosphate for activity. Thus from this point of view the biological role of the phosphorylase b a interconversion may be pictured as the interconversion between two enzymes one of which (phosphorylase b) is in the resting state (i.e., in the absence of ligands) exclusively in the inactive state T whereas the other (phosphorylase a) which catalyzes the same reaction is present to a considerable extent even in the absence of allosteric ligands in the active state R. © 1968, American Chemical Society. All rights reserved.
