Autophosphorylation−Inactivation Site of Hexokinase 2 in Saccharomyces cerevisiae

Abstract

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 in vitro when incubated in the presence of d-xylose [Fernandez et al. (1988) J. Gen. Microbiol. 134, 2493−2498]. This study identifies the site of inactivation by autophosphorylation as serine-158 by observation of a single tryptic peptide difference, peptide sequencing, and size determination by mass spectrometry. Mutation of serine-158 to alanine and cysteine, respectively, prevents autophosphorylation and causes a drastic decrease of the catalytic activity while mutational change to glutamate results in a complete loss of enzyme activity. The catalytically active mutant enzymes display an increased affinity for glucose and exhibit higher K_M with respect to MgATP. Phosphoserine/phosphothreonine-specific protein phosphatase-2A completely reverses the autophosphorylative inactivation of the wild-type enzyme.