Roles of individual enzyme-substrate interactions by alpha-1,3-galactosyltransferase in catalysis and specificity.
Roles of individual enzyme-substrate interactions by alpha-1,3-galactosyltransferase in catalysis and specificity.
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α-1,3-半乳糖基转移酶在催化和特异性中单个酶-底物相互作用的作用。
DOI：
10.1021/bi035430r
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Brew,Keith
中科院分区：
文献类型：
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作者：
Zhang,Yingnan;Swaminathan,GJawahar;Deshpande,Ashlesha;Boix,Ester;Natesh,Ramanathan;Xie,Zhihong;Acharya,KRavi;Brew,Keith
保留糖基转移酶α-1，3-半乳糖基转移酶(α3GT)在人类中突变失活，导致针对其产物α-Gal表位的循环抗体的存在。α3GT催化半乳糖从UDP-Gal转移到β连接的半乳糖苷，如乳糖，在没有受体底物的情况下，催化半乳糖以较低的速率转移到水中。我们使用定点突变来研究α3GT中形成氢键的残基以及与底物的其他相互作用在催化和特异性中的作用。保守的Glu317突变为Gln会削弱乳糖的结合，并使半乳糖转移到乳糖和水的kcat值分别减少2400和120。这种取代并没有干扰结构，但结合的乳糖分子的取向发生了变化。这些变化的幅度不支持先前的假设，即Glu317是双置换机制中的催化亲核剂，表明它作用于受体底物结合，并稳定了半乳糖UDP与C1之间的键断裂的阳离子过渡态。该键的断裂也与α3GT的C-末端区域的构象变化有关，该构象变化与UDP结合相结合。突变实验表明，His280预计与UDP-Gal的半乳糖部分的2-OH相互作用，通过其稳定结合的UDP-Gal在合适的构象中的作用，是严格的供体底物专一性的关键残基。与受体底物形成多重相互作用的Gln247突变为Glu，降低了半乳糖转移到乳糖的催化速率，但不能转移到水中。这种突变被预测会扰乱结合受体底物的取向或环境。这些结果强调了该糖基转移酶中酶和底物之间的氢键在将底物安排在合适的构象和取向以进行有效催化方面的重要性。这些因素表现为催化速率的增加，而不是底物亲和力的增加。
The retaining glycosyltransferase, α-1,3-galactosyltransferase (α3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the α-Gal epitope. α3GT catalyzes galactose transfer from UDP-Gal to β-linked galactosides, such as lactose, and in the absence of an acceptor substrate, to water at a lower rate. We have used site-directed mutagenesis to investigate the roles in catalysis and specificity of residues in α3GT that form H-bonds as well as other interactions with substrates. Mutation of the conserved Glu317to Gln weakens lactose binding and reduces thekcatfor galactosyltransfer to lactose and water by 2400 and 120, respectively. The structure is not perturbed by this substitution, but the orientation of the bound lactose molecule is changed. The magnitude of these changes does not support a previous proposal that Glu317is the catalytic nucleophile in a double displacement mechanism and suggests it acts in acceptor substrate binding and in stabilizing a cationic transition state for cleavage of the bond between UDP and C1 of the galactose. Cleavage of this bond also linked to a conformational change in the C-terminal region of α3GT that is coupled with UDP binding. Mutagenesis indicates that His280, which is projected to interact with the 2-OH of the galactose moiety of UDP-Gal, is a key residue in the stringent donor substrate specificity through its role in stabilizing the bound UDP-Gal in a suitable conformation for catalysis. Mutation of Gln247, which forms multiple interactions with acceptor substrates, to Glu reduces the catalytic rate of galactose transfer to lactose but not to water. This mutation is predicted to perturb the orientation or environment of the bound acceptor substrate. The results highlight the importance of H-bonds between enzyme and substrates in this glycosyltransferase, in arranging substrates in appropriate conformations and orientation for efficient catalysis. These factors are manifested in increases in catalytic rate rather than substrate affinity.