Zhang Yingnan, Swaminathan G Jawahar, Deshpande Ashlesha, Boix Ester, Natesh Ramanathan, Xie Zhihong, Acharya K Ravi, Brew Keith
Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33341, USA.
Biochemistry. 2003 Nov 25;42(46):13512-21. doi: 10.1021/bi035430r.
The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the alpha-Gal epitope. alpha3GT catalyzes galactose transfer from UDP-Gal to beta-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 alpha3GT that form H-bonds as well as other interactions with substrates. Mutation of the conserved Glu(317) to Gln weakens lactose binding and reduces the k(cat) for 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 Glu(317) is 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 alpha3GT that is coupled with UDP binding. Mutagenesis indicates that His(280), 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 Gln(247), 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.
保留型糖基转移酶α-1,3-半乳糖基转移酶（α3GT）在人类中发生突变失活，导致循环中存在针对其产物α-Gal表位的抗体。α3GT催化半乳糖从UDP-半乳糖转移至β-连接的半乳糖苷，如乳糖，在没有受体底物的情况下，也能以较低速率转移至水。我们利用定点诱变来研究α3GT中形成氢键以及与底物发生其他相互作用的残基在催化和特异性方面的作用。保守的Glu(317)突变为Gln会削弱乳糖结合，并使半乳糖基转移至乳糖和水的k(cat)分别降低2400倍和120倍。这种取代不会干扰结构，但结合的乳糖分子的取向会发生变化。这些变化的程度不支持先前的提议，即Glu(317)是双位移机制中的催化亲核试剂，表明它在受体底物结合以及稳定UDP与半乳糖C1之间键断裂的阳离子过渡态中起作用。该键的断裂还与α3GT C端区域的构象变化相关，该变化与UDP结合相偶联。诱变表明，预计与UDP-半乳糖的半乳糖部分的2-OH相互作用的His(280)，通过其在将结合的UDP-半乳糖稳定在适合催化的构象中的作用，是严格供体底物特异性的关键残基。与受体底物形成多种相互作用的Gln(247)突变为Glu会降低半乳糖转移至乳糖的催化速率，但不会降低转移至水的催化速率。预计这种突变会扰乱结合的受体底物的取向或环境。结果突出了该糖基转移酶中酶与底物之间氢键在将底物排列成适当构象和取向以实现高效催化方面的重要性。这些因素表现为催化速率的增加而非底物亲和力的增加。
