Purification and Subunit Interactions of Yeast Hexokinase

Abstract

A method is described for the purification of two native isoenzymes of yeast hexokinase (A and B) in the presence of phenylmethyl sulphonylfluoride, an inhibitor of yeast proteases. In the absence of this inhibitor two proteolytically modified forms (A′ and B′) were obtained. Investigation of the effects of solvent ionic strength and pH on the dissociation of the native form A and the proteolytically modified form A′ suggests that these enzymes have a common molecular weight and quaternary structure. Both forms exist as dimers of molecular weight 108000 ± 3000 in reversible dissociation equilibrium with monomers of molecular weight 54000 ± 1500. Hexokinase A dissociates to monomer both with increasing ionic strength and in the presence of hexose substrates. Dissociation to constituent polypeptide chains of 54000 molecular weight is effected by maleylation or succinylation of the native enzyme, confirming its dimeric structure. Hexokinase A′ exhibits a pH‐dependent dissociation from dimer to monomer. This dissociation is attributable to two ionising groups on the monomer which are predominantly unionised in the dimer.