Active site lysine promotes catalytic function of pyridoxal 5'-phosphate in alpha-glucan phosphorylases

Tight contact of pyridoxal 5'-phosphate to the substrate phosphate is considered to be a crucial requirement of the phosphorolytic cleavage of polysaccharides by glycogen phosphorylases. This study demonstrates an essential role of lysine 533, the only charged residue in hydrogen bond distance to the phosphate of pyridoxal 5'-phosphate in Escherichia coli maltodextrin phosphorylase. Substitution of Lys533 by Ser reduced the turnover number 600-fold. Addition of monovalent cations significantly increased activity of the Lys533-Ser mutant up to a factor of 10, whereas the apparent affinity for Pi was decreased up to 80-fold. Although substitution of Lys533 by Gln caused a similar reduction of kcat, the Km values remained unchanged, and no response to small cations was observed. These results suggest a key role of the positive charge contributed by Lys533 in catalysis, most probably in maintaining the electrostatic neutrality of the pyridoxal 5'-phosphate and aligning the close phosphate-phosphate contacts indispensable for the proton transfer mechanism.