title: Amino acid sequence of two functional sites in yeast glycogen phosphorylase.
abstract: The structure of two functional sites in baker's yeast (Saccharomyces cerevisiae) glycogen phosphorylase (EC 2.4 1.1) was determined as part of a study on the evolution of regulatory enzymes. S-Carboxymethylated, MaBH4-reduced 32-P-labeled yeast phosphorylase a was cleaved with CNBr, thermolysin, and pepsin. Peptides labeled with 32-P or carrying the fluorescent pyridoxyl marker were isolated and purified using ion-exchange chromatography and gel filtration. CNBr cleavage yielded a single radioactive phosphopeptide (42 residues long) and one small fluorescent peptide with the unique sequence epsilon-Pxy-Lys-Phe-Val-Met. Thermolysin digestion gave rise to one radioactive octapeptide and two fluorescent peptides, 15 and 2 residues long, respectively. From a combination of substractive Edman degradations and digestion with yeast protease C, the sequence of the 32-P-labeled octapeptide was established. Phosphothreonine was identified as the sole phosphorylated amino acid, giving the following structure for the site involved in the covalent regulation of yeast phosphorylase: Leu-Thr(P) -Gly-Phe-Leu-Pro-Gln-Glu. The two fluorescent thermolytic peptides, together with two additional pyridoxyl peptides isolated after peptic digestion of the enzyme yielded the following sequence around the site binding pyridoxal-5'-P, the cofactor essential for phosphorylase activity: Ile-Ser-Thr-Ala-Gly-Thr-Glu-Ala-Ser-Gly-Thr-Ser-Asn-Met-Lys(P Pxy)-Phe-Val-Met. While the phosphorylated site bears no resemblance to the site of covalent control in vertebrate phosphorylases, the pyridoxal-P binding site in the yeast enayme displays remarkable homologies with its animal counterparts; the finding that 14 out of 18 amino acids are identical strongly suggests that the cofactor must be directly involved in catalysis.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001894:Borohydrides; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D006005:Phosphorylases; D011485:Protein Binding; D011730:Pyridoxal; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013820:Thermolysin
chemical_list: D000596:Amino Acids; D001894:Borohydrides; D010446:Peptide Fragments; D011730:Pyridoxal; D006005:Phosphorylases; D010447:Peptide Hydrolases; D010434:Pepsin A; D013820:Thermolysin; D003488:Cyanogen Bromide