Glycosynthase activity of Bacillus licheniformis 1,3-1,4-β-glucanase mutants:: Specificity, kinetics, and mechanism
Glycosynthase activity of Bacillus licheniformis 1,3-1,4-β-glucanase mutants:: Specificity, kinetics, and mechanism
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DOI：
10.1021/bi030131n
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发表时间：
2003-11-18
期刊：
影响因子：
2.9
通讯作者：
Planas, A
中科院分区：
文献类型：
--
作者：
Faijes, M;Pérez, X;Planas, A
工程化的糖苷酶保留缺乏水解酶活性的糖苷酶，其有效催化转糖基化反应。用地衣芽孢杆菌1，3 - 1，4-β-葡聚糖酶的E134 A突变体探讨了糖合酶反应的机制。这种内切糖合酶具有区域特异性，可与α-糖基氟化物供体（昆布糖基作为最小供体）和在非还原端含有葡萄糖或木糖的寡糖受体（芳基单糖或寡糖）形成β-1，4-糖苷键。糖合酶活性的pH依赖性反映了一般的碱催化作用，动力学pK（a）为5.2+/-0.1。水溶性碳二亚胺（EDC）的酶失活动力学与活性位点羧酸基团的修饰一致，pK（a）为5.3+/-0.2。一般碱基是Glul 38（在亲本野生型酶中充当一般酸碱的残基），如通过制备双突变体E134 A/EI 38 A所探测的。它缺乏糖合酶活性，但使用叠氮化钠作为受体，不需要一般的碱催化，产生了β-糖基叠氮化物产物。与野生型的pK（a）值相比，E134 A糖合酶的Glu 138的pK（a）（k（cat）/K-M上的动力学pK（a）和EDC失活的pK（a））下降了1.8个pH单位，使得相同的残基能够充当糖合酶中的通用碱基。作为供体的Glc β 4Glc β 3GlcalphaF（2）和作为受体的Glc β 4Glc β-pNP（15）之间的E134 A糖苷酶催化的缩合的动力学参数如下：k（cat）= 1.7 s（-1），K-M（受体）= 11 mM，和K-M（供体）<0.3mM。对缩合和延伸反应进行动力学评价，以建立糖合酶反应在寡糖合成中的制备性应用的条件。芳基单糖和纤维二糖苷受体的产率为70-90%，但海带二糖苷的产率为25-55%，较低的产率（和较低的初始速率）是由于β-1，3-连接的二糖受体对酶的供体亚位点的竞争性抑制。
Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficiently catalyze transglycosylation reactions. The mechanism of the glycosynthase reaction is probed with the E134A mutant of Bacillus licheniformis 1,3-1,4-beta-glucanase. This endo-glycosynthase is regiospecific for formation of a beta-1,4-glycosidic bond with alpha-glycosyl fluoride donors (laminaribiosyl as the minimal donor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (aryl monosaccharides or oligosaccharides). The pH dependence of the glycosynthase activity reflects general base catalysis with a kinetic pK(a) of 5.2+/-0.1. Kinetics of enzyme inactivation by a water-soluble carbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pK(a) of 5.3+/-0.2. The general base is Glul38 (the residue acting as the general acid-base in the parental wildtype enzyme) as probed by preparing the double mutant E134A/EI38A. It is devoid of glycosynthase activity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a beta-glycosyl azide product. The pK(a) of Glu138 (kinetic pK(a) on k(cat)/K-M and pK(a) of EDC inactivation) for the E134A glycosynthase has dropped 1.8 pH units compared to the pK(a) values of the wild type, enabling the same residue to act as a general base in the glycosynthase enzyme. Kinetic parameters of the E134A glycosynthase-catalyzed condensation between Glcbeta4Glcbeta3GlcalphaF (2) as a donor and Glcbeta4Glcbeta-pNP (15) as an acceptor are as follows: k(cat) = 1.7 s(-1), K-M(acceptor) = 11 mM, and K-M(donor) < 0.3 mM. Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions for preparative use of the glycosynthase reaction in oligosaccharide synthesis. Yields are 70-90% with aryl monosaccharide and cellobioside acceptors, but 25-55% with laminaribiosides, the lower yields (and lower initial rates) due to competitive inhibition of the beta-1,3-linked disaccharide acceptor for the donor subsites of the enzyme.