Kinetic and Regulatory Properties of HK I+, a Modified Form of the Type I Isozyme of Mammalian Hexokinase in Which Interactions between the N- and C-Terminal Halves Have Been Disrupted

A modified form (HK I+) of rat Type I hexokinase (HK I) has been expressed. HK I+ contains a centrally located polyalanine insert which, along with the known helical propensity of adjacent sequence, was expected to lead to α-helix formation, with resulting distension of the molecule and disruption of interactions between the N- and C-terminal halves. The properties of HK I+ are consistent with this expectation and with previous proposals that (1) inhibition of HK I by Glc-6-P or its analogs and antagonism of this inhibition by Pi result from competition of these ligands for a binding site in the N-terminal half of HK I, with resulting conformational changes propagated through interactions with the catalytic C-terminal half, and (2) binding of Glc-6-P to a site in the C-terminal half of HK I is obstructed by interactions between the halves, present in HK I but not HK I+.