Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237-->Gln mutant enzymes.
Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237-->Gln mutant enzymes.
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DOI：
10.1042/bj20070042
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发表时间：
2007-05
期刊：
影响因子：
--
通讯作者：
A. Schwarz;L. Brecker;B. Nidetzky
中科院分区：
文献类型：
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作者：
A. Schwarz;L. Brecker;B. Nidetzky
通过定点替换推定的催化Glu 237和使用稳态动力学的纯化的野生型和Glu 237->Gln突变酶的详细比较，研究了肠膜明串珠菌蔗糖磷酸化酶在α-保留葡萄糖基转移的两步酶机制中酸碱催化的作用。在E237 Q中，与需要布朗斯台德催化辅助的底物进行葡萄糖基化或去葡萄糖基化的反应在相应步骤中选择性减慢约10（5）倍。叠氮化物，乙酸盐和甲酸盐，但没有卤化物恢复催化活性高达300倍，在E237 Q的条件下，其中的去葡萄糖基化步骤是速率决定，并促进生产相应的α-葡糖苷。乙酸盐和甲酸盐对E237 Q的化学拯救的原位质子NMR研究表明，酶促形成的α-葡萄糖1-酯通过酰基迁移和水解自发分解。使用kcat/K（m）的pH曲线，分析了野生型和E237 Q的动力学分离的葡糖基化和脱葡糖基化步骤的pH依赖性。野生型的葡萄糖基化分别在约5.6和7.2的表观pK（a）值以上和以下进行，而去葡萄糖基化依赖于一组pK（a）约5.8的表观单电离，其必须被去质子化以进行反应。E237 Q的糖基化减慢至低于表观pK（a）约6.0，但已失去野生型的高pH依赖性。E237 Q的去葡萄糖基化是非pH依赖性的。结果允许明确的分配Glu 237作为蔗糖磷酸化酶的催化酸碱。它们支持一种机制，其中Glu 237的pK（a）在游离酶中约7.2和约葡萄糖基酶中间体中约5.8之间循环，确保谷氨酸残基侧链在催化的每个步骤中的最佳参与。酶去葡糖基化成阴离子亲核试剂发生在Glu 237质子化或未质子化的情况下。结果描绘如何保守的活性位点保留糖苷水解酶组可以容纳磷酸化酶的酶功能。
The role of acid-base catalysis in the two-step enzymatic mechanism of alpha-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237-->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10(5)-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding alpha-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed alpha-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/K(m), the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK(a) values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK(a) approximately 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK(a) approximately 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid-base of sucrose phosphorylase. They support a mechanism in which the pK(a) of Glu237 cycles between approximately 7.2 in free enzyme and approximately 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.