Relaxation spectra of yeast hexokinases. Isomerization of the enzyme

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Abstract
Yeast [baker''s] hexokinase isozymes PI and PII exhibit a pH dependent, rapid relaxation process at 15.degree. C at enzyme concentrations of 100-474 .mu.M and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/.tau. [relaxation] varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for PI and 85 ms-1 for PII at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 .times. 1011 M-1 s-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions, and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. Apparently isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.

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