Yeast Hexokinase: Substrate‐Induced Association‐ Dissociation Reactions in the Binding of Glucose to Hexokinase P‐II

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Abstract
The purification of native hexokinases P-I and P-II from yeast was described using preparative isoelectric focusing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer-dimer association-dissociation reaction were investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupied in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme.cntdot.glucose complex was 3 .times. 10-4 mol l-1 and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I = 0.02 mol l-1, pH < 7.5) was also independent of enzyme concentration (in the range 10-1000 .mu.g ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the 2 available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 .times. 10-3 mol l-1 at pH 7.0 to 6 .times. 10-3 at pH 6.5. Under intermediate conditions (pH 7.0, I = 0.15 mol l-1), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperativity (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of finding the 2 sites to be equivalent is that the dimer results from the homologous association of 2 identical subunits.