Glucose binding isotope effects in the ternary complex of brain hexokinase demonstrate partial relief of ground-state...

Isotope effects can yield detailed information about contacts made between a bound compound and its host receptor or enzyme. In this study, we have measured isotope effects upon the equilibrium constant for the association of glucose and human brain hexokinase [E.C.2.7.1.1] in the presence of beta,gamma-CH(2)-ATP and compared these with data for the same equilibrium in the absence of ATP-analogue. We have found isotope effects of 1.012, 0.929, 1.031, 1.052, 0.998, and 1.032 for the competitive binding of [1-(3)H]-, [2-(3)H]-, [3-(3)H]-, [4-(3)H]-, [5-(3)H]-, or [6,6-(3)H(2)]- and either [2-(14)C]- or [6-(14)C]glucose to brain hexokinase. We observed changes only at H1, H5, and H6, and we attribute these to a slight change in the position of Asn683 and Glu742 due to nucleotide binding and to partial satisfaction of activated OH6 by the terminal nucleotide phosphorus.