Purification and crystallization of glycogen phosphorylase from Saccharomyces cerevisiae.
Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein. We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts. To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme. To this end, we have purified, characterized and crystallized glycogen phosphorylase from S. cerevisiae. The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter. One liter of cultured cells yields 12 mg of crystallizable material. The purified protein was not phosphorylated and had an activity of 4.7 units/mg in the presence of saturating amounts of substrate. Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution. The latter belongs to space group P4(1)2(1)2 with unit cell constants of a = 161.1 A and c = 175.5 A Based on the density of the crystals, the solvent content is 49.7%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase.
Rath
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VL
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Hwang
P K
PK
Fletterick
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010766:Phosphorylation; D010957:Plasmids; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D006005:Phosphorylases