Potentiometry, calorimetry, and spectrophotometry have been used to analyze the coupling between the binding of protons and substrate analogues to Escherichia coli aspartate transcarbamylase (c6r6) and its catalytic subunit (c3). Changes in the states of ionization of groups on the protein which accompany ligand binding are shown to make a substantial contribution to the overall energetics of ligand binding and also to provide a possible basis for some of the fundamental differences between c3 and c6r6. Over the pH range 6.5-10.2, binding of the bisubstrate analogue N-phosphonoacetyl-l-aspartate (PALA) to both c6r6 and c3 is accompanied by the absorption of protons. The pH profile AΔ$$H+ for c3 is bell-shaped, with a maximum at pH 8.8, while the curve for c6r6 has a broad maximum extending from pH 7.5 to 9.5. PALA linked proton binding is noncooperative in 0.05 M NaOAc at both pH 7 and 8.3. Protons are also absorbed when succinate binds to c6r6; however, the variation in AΔ$$H+ with pH is small. In contrast, pH profiles of AΔ$$H+ for the binding of carbamyl phosphate to both c6r6 and c3 have two well-defined maxima at pH ~6.5 and ~9.5. The carbamyl phosphate data have been interpreted in terms of increases in the pK values of three groups in c6r6 and two groups in c3 upon binding. The two groups titrating below pH 8 in c6r6 are estimated to have pK values of 6.3 and ΔHionization = 5.8 ± 0.8 kcal/mol; for the group titrating above pH 8, PK ~ 9.1 and ΔHionization ~ 10 kcal/mol. The corresponding pK values for c3 are 5.5 and 9.5. The proton effects associated with the binding of succinate are in fair agreement with a model proposed previously [Beard, C. B., & Schmidt, P. D. (1973) Biochemistry 12, 2255-2264], which implicates a group with a pK value of 8.3 in binding and a group with pK ~ 6.2 in a conformation transition. However, evidence for the possible involvement of a group with pK ~ 7.5 and ΔHionization ~ 5.5 kcal/mol is also presented. Comparison of the pH dependence of Δ$$H+ for the three ligands strongly suggests that the phosphonate group of bound PALA is fully ionized. The effects of oxidizing the active-site cysteine [Vanaman, T. C., & Stark, G. R. (1970) J. Biol. Chem. 245, 3565-3573; Benisek, W. F. (1971) J. Biol. Chem. 246, 3151-3159] are consistent with this conclusion. They also suggest that this residue interacts with both bound carbamyl phosphate and bound PALA. © 1979, American Chemical Society. All rights reserved.
