In vivo phosphorylation and in vitro autophosphorylation-inactivation of Kluyveromyces lactis hexokinase KlHxk1

• The bifunctional hexokinase KlHxk1 is phosphorylated in vivo at serine-15.
• KlHxk1 is partially inactivated in vitro by autophosphorylation at serine-157.
• Crystallized phosphoserine-15 KlHxk1 exhibits a symmetrical dimeric structure.
• Phosphorylation at serine-15 destabilizes the KlHxk1 homodimer in solution.
• KlHxk1 phosphorylation at serine-15 presumably contributes to glucose signalling.

The bifunctional hexokinase KlHxk1 is a key component of glucose-dependent signal transduction in Kluyveromyces lactis. KlHxk1 is phosphorylated in vivo and undergoes ATP-dependent autophosphorylation-inactivation in vitro. This study identifies serine-15 as the site of in vivo phosphorylation and serine-157 as the autophosphorylation-inactivation site. X-ray crystallography of the in vivo phosphorylated enzyme indicates the existence of a ring-shaped symmetrical homodimer carrying two phosphoserine-15 residues. In contrast, small-angle X-ray scattering and equilibrium sedimentation analyses reveal the existence of monomeric phosphoserine-15 KlHxk1 in solution. While phosphorylation at serine-15 and concomitant homodimer dissociation are likely to be involved in glucose signalling, mechanism and putative physiological significance of KlHxk1 inactivation by autophosphorylation at serine-157 remain to be established.