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)
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F.
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Ramírez-Silva
M T
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de Gómez-Puyou
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Gómez-Puyou
D000818:Animals; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014867:Water / Q000494:pharmacology*
D008823:Micelles; D014867:Water; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D008274:Magnesium; D011188:Potassium