The Modification of Yeast Hexokinases by Proteases and Its Relationship to the Dissociation of Hexokinase into Subunits

Crystalline hexokinase prepared by the method of Darrow and Colowick consists largely of enzyme which has been modified in its chromatographic and electrophoretic behavior by yeast protease which is present during the isolation procedure. This proteolytic modification can be prevented by removal of the protease by chromatography on diethylaminoethyl cellulose; it can also be largely inhibited by phenylmethanesulfonyl fluoride. Preparations made by the diethylaminoethyl cellulose method contain two forms of the enzyme (P-I and P-II), both of which are more basic by chromatography and electrophoresis than is protease-modified enzyme (S forms). Form P-II shows the same catalytic activity as the Darrow-Colowick preparation, but Form P-I is less active. The two forms are equally susceptible to modification by trypsin. Forms P-I and P-II exist mainly as tetramers of molecular weight about 100,000 at low ionic strength in the neutral pH region, as measured by ultracentrifuge, light scattering, or gel filtration. They tend to dissociate, without loss of catalytic activity, to dimers as the ionic strength, temperature, or pH is raised or when glucose is added in the presence of phosphate. They can be converted to S forms by trypsin or yeast protease only when glucose or salts are present, suggesting that the enzyme must be in the dimer state in order for modification to occur. The protease-modified enzyme tends to exist mainly as a dimer of molecular weight around 50,000 at neutral pH. Thus, modification by trypsin seems to remove some groups which normally play a role in the association of dimer molecules to form the tetramer structure. Mercaptoethanol causes a striking decrease in sedimentation rate when added to the enzyme at pH 9.0. This may be correlated with the recent finding that mercaptoethanol can cause inactivation of the enzyme at this pH.