Fluorescence-quenching study of glucose binding by yeast hexokinase isoenzymes

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Abstract
A study of the effect of varying ionic strength on the glucose-induced quenching of tryptophan fluorescence of hexokinase isoenzymes A(P-I) and B(P-II) was carried out at pH 8.3 and 5.5. At pH 8.3 both isoenzymes gave apparently linear Scatchard-type data plots even with protein concentrations and ionic strengths for which both dimeric and monomeric forms of hexokinase coexist in significant amounts. Taking into account a 1% accuracy in the experimental measurements, it was concluded that the intrinsic dissociation constants, Km and Kd, for the binding of glucose to the monomeric and dimeric forms of HkB, are within a factor of 2 of each other, i.e., Kd/Km .ltoreq. 2. The values of Km, estimated from the apparent K, were so greatly influenced by ionic strength that it is apparently meaningless to compare Km and Kd values measured at different ionic strengths as was done in the literature. Curvature in the pH 5.5 fluorescence-quenching plots for relatively low ionic strengths demonstrates cooperativity for glucose-binding to the dimer, positive for HkA but negative for HkB. The binding is relatively non-cooperative at high ionic strength at this pH. These results were attributed to the well known effect of salt-neutralization of side chain electrical charges on the flexibility and compactness of proteins.