Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg²⁺, Mn²⁺, Ca²⁺, Cd²⁺, and Zn²⁺ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20°C. At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn²⁺ or Ca²⁺ was present in the buffered isozyme solution before the glucose titration, whereas Mg²⁺ caused the glucose binding to become noncooperative. Zn²⁺ and Cd²⁺ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4%. Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II. Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, as well as in buffer alone, at both pH's. At the lower pH, Ca²⁺ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn²⁺ increased it only about 40% and Mg²⁺ not at all. Further, Ca²⁺ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg²⁺ and Mn²⁺ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase. However, at pH 6.5 the Ca²⁺ effect on the cooperativity was negligible, while Mg²⁺ and Mn²⁺ decreased the coefficient from 1.6 in buffer to about 1.4. The biological implications of these diverse metal ion effects are discussed.