tailieunhanh - Báo cáo khoa học: 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 selec-tivity of pyruvate kinase in water/dimethylsulfoxide mix-tures by measuring the activation and inhibition constants of K + and Na + , . their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respect-ively [Melchoir . (1965) Biochemistry4, 1518–1525]. In 40% dimethylsulfoxide appfor K + and Na + were 190 and 64-fold lower than in K + and Na +decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Kiapp/ app for K + and Na + were 34– and –, respectively | Eur. J. Biochem. 270 2377-2385 2003 FEBS 2003 doi Selectivity of pyruvate kinase for Na and K in water dimethylsulfoxide mixtures Leticia Ramirez-Silva and Jesus Oria-Hernandez Departamento de Bioquimica Facultad de Medicina Universidad Nacional Autonoma de Mexico Mexico 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 . their binding to the monovalent and divalent cation binding sites of pyruvate kinase respectively Melchoir . 1965 Biochemistry 4 1518-1525 . In 40 dimethylsulfoxide the 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 app for K and Na were and 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 . Keywords dimethylsulfoxide magnesium ion potassium ion pyruvate kinase sodium ion. Rabbit muscle pyruvate kinase .