Water-induced transitions in the K+ requirements for the activity of pyruvate kinase entrapped in reverse micelles.

The activity of pyruvate kinase was studied in reverse micelles formed with cetyltrimethylammonium bromide, n-octane, hexanol, and various amounts of water. In systems with 100% water, K+ is an essential activator of pyruvate kinase [Kachmar, J. F., & Boyer, P. D. (1953) J. Biol. Chem. 200, 669-683]; i.e., without and with K+, the activities observed were 0.07 and 300 mumol/(min.mg), respectively. In the micellar system with 3.6% water (v/v), pyruvate kinase exhibited an activity of about 45 mumol/(min.mg), in the absence of K+. The kcat was about 450 times larger than that in 100% water without K+. Km values for ADP and phosphoenolpyruvate differed, but not markedly from those in 100% water with or without K+. The kinetics of pyruvate kinase in reverse micelles were not affected by K+. The activity curve of pyruvate kinase in reverse micelles without K+ in a pH range of 6.0-8.5 was almost superimposable to that of the enzyme in 100% water with K+, and it differed drastically from that in 100% water without K+. The fluorescence emission spectra of pyruvate kinase in 100% water exhibited a blue shift of 3 nm upon the addition of ligands (Mg2+, phosphoenolpyruvate, and K+) that cause a transition of the enzyme to its active state. Without ligands, the entrapment of pyruvate kinase in reverse micelles with 3.0% water produced a blue shift of nearly 2 nm with respect to that of the enzyme in 100% water without ligands. As water was raised to 7.0% (v/v), the maximal emission shifted to longer wavelengths; these changes paralleled the appearance of the K(+)-dependent activity.(ABSTRACT TRUNCATED AT 250 WORDS)