Association equilibria and reacting enzyme gel filtration of yeast hexokinase.

The association-dissociation of yeast hexokinase has been re-examined and the size of the reacting form of the enzyme has been investigated under a variety of conditions. Both Sephacryl S-200 and high performance liquid chromatography on Bio-Sil have been employed with continuous effluent monitoring under reacting and nonreacting conditions. The reacting enzyme was monomeric under all conditions, suggesting that the dimer is not an important catalytic species in normal assays. The reacting enzyme remained as a monomer under the following conditions: 0.1-15 micrograms/ml loading concentration, from 30 to 100 mM ionic strength, with 2 mM citrate, with 50% D2O, and at 160 atm hydrostatic pressure. The dissociation constant for the nonreacting hexokinase was 22 (+/- 2) micrograms/ml (uncorrected for 5-fold dilution) in 100 mM triethanolamine, pH 6.5, and 25 degrees C. Glucose or MgATP had dissociative effects under all conditions studied, but MgATP was much less effective and only slightly more effective than an equivalent ionic strength. NaCl, between 30 and 80 mM, promoted dissociation, with a concomitant conformational change suggested by nonlinearity of log-log plots. The extent of dissociation with MgCl2 was slightly greater than an equivalent NaCl ionic strength and the shape of the association curves suggested the formation of an elongated dimer in the presence of MgCl2. The conclusion is made that hexokinase is monomeric under most assay conditions and that the dissociation is predominantly the result of the glucose interaction. High performance liquid chromatography has been shown to be a useful method of assessing the association state of enzymes under both reacting and nonreacting conditions.