Schinzel R
Department of Physiological Chemistry, University of Würzburg Biozentrum, Federal Republic of Germany.
J Biol Chem. 1991 May 25;266(15):9428-31.
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.
磷酸吡哆醛与底物磷酸紧密接触被认为是糖原磷酸化酶对多糖进行磷酸解裂解的关键要求。本研究证明了赖氨酸533的重要作用，它是大肠杆菌麦芽糊精磷酸化酶中与磷酸吡哆醛的磷酸基团处于氢键距离的唯一带电荷残基。用丝氨酸取代赖氨酸533使周转数降低了600倍。添加单价阳离子可使赖氨酸533 - 丝氨酸突变体的活性显著提高达10倍，而对无机磷酸（Pi）的表观亲和力降低达80倍。尽管用谷氨酰胺取代赖氨酸533导致催化常数（kcat）有类似程度的降低，但米氏常数（Km）值保持不变，并且未观察到对小阳离子的响应。这些结果表明赖氨酸533所贡献的正电荷在催化中起关键作用，很可能是在维持磷酸吡哆醛的静电中性以及使质子转移机制所必需的紧密磷酸 - 磷酸接触排列整齐方面发挥作用。
