The Binding of Glucose to Native and Proteolytically Modified Yeast Hexokinase PI

The binding of glucose to native, dimeric yeast hexokinase PI and to its proteolytically modified product, the monomeric SI form, has been investigated by equilibrium dialysis and by titration of the quenching of intrinsic protein fluorescence. The effects of glucose concentration, pH and ionic strength on binding have been examined and the way in which these factors also affect the monomer‐dimer association‐dissociation reaction of the native‐enzyme has been investigated by analytical ultracentrifugation. At pH 6.0 and pH 8.5 the dissociation constants of glucose from hexokinase SI monomer were 0.11 mmoll−1 and 0.23 mmol 1−1 respectively and each was independent of ionic strength over the range 0–1.0 mol KCl 1−1. The dependence of the dissociation constant of glucose from SI on pH showed a sharp transition between these two extremes over a narrow interval from pH 7 to pH 7.5 At pH 8 the affinity of glucose for proteolytically modified SI monomer was the same as that for native PI monomer formed in the presence of 1.0 mol KCll−1 in support of the idea that the former is a good analogue of the latter. At pH 6.7 and low ionic strength, the binding of glucose to hexokinase PI dimer alone shows strong positive cooperativity. This behaviour is in marked contrast to that of the PII isoenzyme for which both binding sites on the dimer are equivalent in solution and binding is non‐cooperative. At pH 6.7, the quenching of intrinsic protein fluorescence as a function of glucose concentration (Hill coefficient, h= 1.3) markedly precedes binding (h= 1.6) to hexokinase PI, the concentration at half‐saturation for each being 0.21 mmol l−1 and 0.82 mmol l−1 respectively. Detailed analysis of the data according to the Adair equation is presented. At pH 8.0, hexokinase PI exists in association‐dissociation equilibrium between monomer and dimer. At low ionic strength, despite the presence of some monomer which binds glucose more strongly than dimer, the affinity of hexokinase PI is markedly reduced at pH 8.0 compared with that at pH 6.7 where it existed solely as dimer. It is concluded from linkage calculations that the instrinsic affinity of the dimer for glucose decreases by the same factor of two as does the monomer over this pH range and that the binding of glucose to PI dimer is non‐cooperative at pH 8.0. There is thus a structural transition from a cooperative to a non‐cooperative dimer, possibly occurring over the same narrow interval from pH 7 to 7.5 as the change in affinity of the monomer. pH is a major factor in determining the relative properties of hexokinases PI and PII. The ratio of the affinity of the monomer for glucose to that of the dimer is about six and is independent of pH above pH 6.7. Under conditions where hexokinase PI exists as a mixture of monomers and dimers, changes in the binding of glucose as a function of ionic strength can be accounted for entirely by changes in the monomer‐dimer equilibrium.