AUTOPHOSPHORYLATION-INACTIVATION SITE OF HEXOKINASE-2 IN SACCHAROMYCES-CEREVISIAE

Hexokinase 2 from Saccharomyces cerevisiae is phosphorylated in vivo a t serine-15 [Kriegel et al. (1994) Biochemistry 33, 148-152] and under goes ATP-dependent autophosphorylation-inactivation in vitro when incu bated in the presence of D-xylose [Fernandez et al. (1988) J. Gen. Mic robiol. 134, 2493-2498]. This study identifies the site of inactivatio n by autophosphorylation as serine-158 by observation of a single tryp tic peptide difference, peptide sequencing, and size determination by mass spectrometry. Mutation of serine-158 to alanine and cysteine, res pectively, 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 mutan t enzymes display an increased affinity for glucose and exhibit higher K-M with respect to MgATP. Phosphoserine/phosphothreonine-specific pr otein phosphatase-2A completely reverses the autophosphorylative inact ivation of the wild-type enzyme.