Dimethylsulfoxide promotes K+-independent activity of pyruvate kinase and the acquisition of the active catalytic conformation.
Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramírez-Silva, L., Tuena de Gómez-Puyou, M., & Gómez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 micromol x min(-1) x mg(-1) in 40% dimethylsulfoxide, compared with 280 and 0.023 micromol x min(1) x mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg(2+), phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30-40% dimethylsulfoxide coincided with that of the enzyme-Mg(2+)-phosphenolpyruvate-K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase-Mn(2+)-(CH3)4N+ complex in 30-40% dimethylsulfoxide were similar to those of the pyruvate kinase-Mn(2+)-K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces its active conformation.
Ramírez-Silva
L
L
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F., México. lramirez@laguna.fmedic.unam.mx
Ferreira
S T
ST
Nowak
T
T
Tuena de Gómez-Puyou
M
M
Gómez-Puyou
A
A
D001665:Binding Sites; D002384:Catalysis; D002414:Cations, Monovalent; D004121:Dimethyl Sulfoxide / Q000737:chemistry* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation / Q000187:drug effects; D011770:Pyruvate Kinase / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D014867:Water / Q000737:chemistry
D002414:Cations, Monovalent; D012997:Solvents; D014867:Water; D011770:Pyruvate Kinase; D011188:Potassium; D004121:Dimethyl Sulfoxide