Faijes Magda, Pérez Xavi, Pérez Odette, Planas Antoni
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.
Biochemistry. 2003 Nov 18;42(45):13304-18. doi: 10.1021/bi030131n.
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 Glu138 (the residue acting as the general acid-base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A. 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.
糖基合酶是经过改造的保留型糖苷酶，缺乏水解酶活性，能有效催化转糖基化反应。利用地衣芽孢杆菌1,3 - 1,4-β-葡聚糖酶的E134A突变体探究了糖基合酶反应的机制。这种内切糖基合酶在与α-糖基氟供体（以层二糖基作为最小供体）以及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-糖苷键时具有区域特异性。糖基合酶活性的pH依赖性反映了一般碱催化作用，其动力学pK(a)为5.2±0.1。水溶性碳二亚胺（EDC）使酶失活的动力学与一个pK(a)为5.3±0.2的活性位点羧酸盐基团的修饰相一致。通过制备双突变体E134A/E138A探究发现，一般碱是Glu138（在亲本野生型酶中作为一般酸碱的残基）。它没有糖基合酶活性，但使用叠氮化钠作为不需要一般碱催化的受体时会产生β-糖基叠氮产物。与野生型的pK(a)值相比，E134A糖基合酶的Glu138的pK(a)（关于k(cat)/K(M)的动力学pK(a)和EDC失活的pK(a)）下降了1.8个pH单位，使得同一个残基能够在糖基合酶中充当一般碱。以Glcbeta4Glcbeta3GlcalphaF(2)作为供体和Glcbeta4Glcbeta-pNP(15)作为受体时，E134A糖基合酶催化缩合反应的动力学参数如下：k(cat)=1.7 s(-1)，K(M)(受体)=11 mM，K(M)(供体)<0.3 mM。对供体的自缩合和延伸反应进行动力学评估，以确定在寡糖合成中制备性使用糖基合酶反应的条件。对于芳基单糖和纤维二糖苷受体，产率为70 - 90%，但对于层二糖苷，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位点的竞争性抑制。
