Selectivity of pyruvate kinase for Na + and K + in water/dimethylsulfoxide mixtures

In aqueous media, muscle pyruvate kinase is highly selective for K + over Na + . We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K + and Na + , i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4 , 1518–1525]. In 40% dimethylsulfoxide the K 0.5 app for K + and Na + were 190 and 64‐fold lower than in water. K i app for K + and Na + decreased 116 and 135‐fold between 20 and 40% dimethylsulfoxide. The ratios of K i app / K 0.5 app for K + and Na + were 34–3.5 and 3.3–0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K + and Na + into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K + and Mg 2+ exhibit high selectivity for their respective cation binding sites, whereas when Na + substitutes K + , Na + and Mg 2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg 2+ and K + for the monovalent cation binding site, which is close to 200. For Na + and Mg 2+ this ratio is approximately 20. Therefore, the data suggest that K + induces conformational changes that prevent the binding of Mg 2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K + and Na + indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K + and Na + .