Autophosphorylation-inactivation site of hexokinase 2 in Saccharomyces cerevisiae.
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.
Heidrich
K
K
Technische Universität Dresden, Medizinische Fakultät Carl Gustav Carus, Institut für Physiologische Chemie, Germany.
Otto
A
A
Behlke
J
J
Rush
J
J
Wenzel
K W
KW
Kriegel
T
T
D000409:Alanine / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000235:genetics
D012694:Serine; D006593:Hexokinase; D003545:Cysteine; D000409:Alanine