Amino‐Acid Sequence of the Pyridoxal‐Phosphate‐Binding Site in Escherichia coli Maltodextrin Phosphorylase

The primary sequence of the pyridoxal 5′‐phosphate binding site in Escherichia coli maltodextrin phosphorylase was determined as part of a study on the role of pyridoxal phosphate for structure and function of phylogenetically different phosphorylases with different regulatory properties. Maltodextrin phosphorylase was isolated and purified by an improved procedure. The enzyme contains 2 mol pyridoxal phosphate per mol of dimer in accordance with other phosphorylases. The sodiumborohydride‐reduced phosphorylase was cleaved by Staphyllococcus aureus protease and a peptide carrying the fluorescent pyridoxyl marker was isolated by gel filtration and differential ion‐exchange chromatography before and after phosphatase treatment. A sequence, 21 residues long, was established partially by automatic Edman degradation and from overlap of proteolytic fragments as: Ala‐Ser‐Gly‐Thr‐Gly‐Asn‐Met‐Lys(PPxy)‐Leu‐Ala‐Leu‐Asp‐Gly‐Ala‐Leu‐Thr‐Val‐Gly‐Thr‐Leu‐Asp. The part of the sequence preceding Lys(PPxy), N6‐(phosphopyridoxyl)‐l‐lysine, is identical with the corresponding sequence in rabbit muscle phosphorylase. Among the five amino acid exchanges observed distal to the cofactor binding site, four can be ascribed to conservative substitutions. A comparison of all known sequences of pyridoxal phosphate binding sites in phosphorylases shows a region of high mutability among the four amino acid residues immediately following the cofactor binding site.