Calorimetric determination of thermodynamic parameters of reaction reveals different enthalpic compensations of the yeast hexokinase isozymes

The change in enthalpy and rate constants for the reactions of yeast hexokinase isozymes, PI (Hxk1) and PII (Hxk2), was determined at pH 7.6 and 25 degreesC by isothermal titration calorimetry. The reactions were done in five buffer systems with enthalpy of protonation varying from -1.22 kcal/mol ( phosphate) to -11.51 kcal/mol ( Tris), allowing the determination of the number of protons released during glucose phosphorylation. The reaction is exothermic for both isozymes with a small, but significant ( p < 0.0001), difference in the enthalpy of reaction (Delta H-R), with an Delta H-R of -5.1 +/- 0.2 ( mean +/- S. D.) kcal/mol for Hxk1, and an Delta H-R of -3.3 +/- 0.3 (mean +/- S. D.) kcal/mol for Hxk2. The K-m for ATP determined by ITC was very similar to those reported in the literature for both isozymes. The effect of NaCl and KCl, from 0 to 200 mM, showed that although the rate of reaction decreases with increasing ionic strength, no change in the Delta H-R was observed suggesting an entropic nature for the ionic strength. The differences in Delta H-R obtained here for both isozymes strongly suggest that, besides glucose phosphorylation, another side reaction such as ATP hydrolysis and/or enzyme phosphorylation is taking place.