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 K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 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 Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ 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+.
Ramírez-Silva
Leticia
L
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, México. lramirez@laguna.fmedic.unam.mx
Oria-Hernández
Jesús
J
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002412:Cations; D002942:Circular Dichroism; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry*; D011817:Rabbits; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013816:Thermodynamics; D014466:Ultraviolet Rays; D014867:Water / Q000737:chemistry
D002412:Cations; D007477:Ions; D014867:Water; D012964:Sodium; D011770:Pyruvate Kinase; D008274:Magnesium; D011188:Potassium; D004121:Dimethyl Sulfoxide