Amino acid sequence of two functional sites in yeast glycogen phosphorylase.
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.
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
D000596:Amino Acids; D001894:Borohydrides; D010446:Peptide Fragments; D011730:Pyridoxal; D006005:Phosphorylases; D010447:Peptide Hydrolases; D010434:Pepsin A; D013820:Thermolysin; D003488:Cyanogen Bromide