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, EJ. (1971) Arch. Biochem. Bioph ys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles e xhibits an important K+-independent activity [Ramirez-Silva,L., Tuena de Go mez-Puyou,M., & Gomez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is p ossible that the amount of water, as well as interactions of the protein wi th the micelles, can account for this behavior. We therefore explored the s olvent effects on the catalytic properties of muscle pyruvate kinase. The e nzyme exhibited an activity of 19.4 mu mol min(-1). mg(-1) in 40% dimethyls ulfoxide, compared with 280 and 0.023 mu mol. min(-1).mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic con stants for the substrates of pyruvate kinase in dimethylsulfoxide without K + were similar to those in 100% water with K+, and differed from those in w ater without K+. The spectral center of mass of the emission spectrum of py ruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presenc e of Mg2+, phosphenolpyruvate, and K+, ligands that induce the active confo rmation of the enzyme. The spectral center of mass of the apoenzyme in 30-4 0% dimethylsulfoxide coincided with that of the enzyme -Mg2+ -phosphenolpyr uvate-K+ complex in 100% water. The water relaxation rate enhancement facto r and binding of phosphenolpyruvate to the pyruvate kinase-Mn2+-(CH3)(4)Ncomplex in 30-40% dimethylsulfoxide were similar to those of the pyruvate k inase-Mn2+-K+ complex in water. The aforementioned results indicate that wh en muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces i ts active conformation.