Activation of Yeast Pyruvate Kinase by Natural and Artificial Cryoprotectants

The inactivation of yeast pyruvate kinase at temperatures near 0° and the stabilization achieved by addition of 50% glycerol or 0.1 m KCl and 24 mm MgCl2 recently documented (Kuczenski, R. T., and Suelter, C. H., Biochemistry, 9, 939, 1970) prompted an examination of the effects of glycerol and other cryoprotectants on the kinetic activity of this enzyme. We now report that addition of glycerol, dimethyl sulfoxide, trimethylphosphine oxide, or dextran to an assay mixture containing less than Km levels of substrates activates yeast pyruvate kinase often to the same extent as that observed by addition of the positive modifier, fructose 1,6-diphosphate, in the absence of the cryoprotectant. Maximum effects were observed with 33% glycerol, 20% dimethyl sulfoxide, and 14% trimethylphosphine oxide. The activation by glycerol and dimethyl sulfoxide at 30° results from a lowering of the Km for phosphoenolpyruvate from 1.1 mm to 0.3 to 0.4 mm and the Hill slope (nh) from 3.1 to 2.1. Kinetic analysis at 0° in the absence of the cryoprotectant gave a Km of 0.13 mm and nh = 0.94. These changes in the kinetics of yeast pyruvate kinase are discussed in terms of the effect of temperature and solvent on the structure of both water and enzyme.