Autophosphorylation−Inactivation Site of Hexokinase 2 in Saccharomyces cerevisiae†

摘要: Hexokinase 2 from Saccharomyces cerevisiae is phosphorylated in vivo at serine-15 [Kriegel et al. (1994) Biochemistry 33, 148-152] and undergoes ATP-dependent autophosphorylation-inactivation vitro when incubated the presence of D-xylose [Fernandez (1988) J. Gen. Microbiol. 134, 2493-2498]. This study identifies site inactivation by autophosphorylation as serine-158 observation a single tryptic peptide difference, sequencing, size determination mass spectrometry. Mutation to alanine cysteine, respectively, prevents causes drastic decrease catalytic activity while mutational change glutamate results complete loss enzyme activity. The catalytically active mutant enzymes display an increased affinity for glucose exhibit higher K(M) with respect MgATP. Phosphoserine/phosphothreonine-specific protein phosphatase-2A completely reverses autophosphorylative wild-type enzyme.