IN-VIVO PHOSPHORYLATION SITE OF HEXOKINASE-2 IN SACCHAROMYCES-CEREVISIAE

Yeast hexokinase 2 is known to be a phosphoprotein in vivo, prominentl y labeled from P-32-inorganic phosphate after a shift of cells to medi um with low glucose concentration [Vojtek, A. B., & Fraenkel D.G. (199 0) Eur. J. Biochem. 190, 371-375]. The principal and perhaps sole site of phosphorylation is now identified as residue serine-15, by observa tion of a single tryptic peptide difference, its sequencing and size d etermination by mass spectrometry, and by mutation to alanine, which p revents phosphorylation in vivo. Although protein kinase A was unlikel y to accomplish the phosphorylation in vivo, serine-15 does belong to a protein kinase A consensus phosphorylation sequence, and in vitro ph osphorylation by protein kinase A at serine-15 could be shown by label ing and by peptide determination. The alanine-15 mutant enzyme was not phosphorylated in vitro.