α-Lactalbumin (LA) stimulates milk β-1,4-galactosyltransferase I (β4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine -: Crystal structure of β4Gal-T1-LA complex with UDP-Glc
α-Lactalbumin (LA) stimulates milk β-1,4-galactosyltransferase I (β4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine -: Crystal structure of β4Gal-T1-LA complex with UDP-Glc
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DOI：
10.1074/jbc.m102458200
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发表时间：
2001-10-05
影响因子：
4.8
通讯作者：
Qasba, PK
中科院分区：
文献类型：
--
作者：
Ramakrishnan, B;Shah, PS;Qasba, PK
β-1，4-半乳糖基转移酶1（Gal-T1）将半乳糖（Gal）从UDP-Gal转移到N-乙酰葡糖胺（Glc-NAc），这构成其正常的半乳糖基转移酶（Gal-T）活性。在α-乳白蛋白（IA）存在下，它将Gal转化为Glc，这是它的乳糖合成酶（LS）活性。它还将葡萄糖（Glc）从UDP-Glc转移到GlcNAc，构成葡糖基转移酶（Glc-T）活性，尽管效率仅为Gal-T活性的0.3-0.4%。在本研究中，我们发现LA使这种活性增加了近30倍。它还增强了对各种N-酰基取代的葡萄糖胺受体的Glc-T活性。Glc-T反应的稳态动力学研究表明，在不存在LA的情况下，供体和受体底物的Km值较高。在LA的存在下，受体底物的Km降低30倍，而UDP-Glc仅降低5倍。为了理解该性质，我们已经确定了Gal-TI-IA与UDP-Glc·Mn 2+和与N-丁酰基-葡糖胺（N-丁酰基-GlcN）（Glc-T活性中的优选糖受体）的复合物的晶体结构。晶体结构表明，尽管UDP-Glc的结合与UDP-Gal非常相似，但在UDP-Glc和Gal-T1之间的氢键相互作用中观察到很少的显著差异。基于目前的动力学和晶体结构的研究，LA在Glc-T活性中的作用的一个可能的解释已被提出。
beta -1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (Glc-NAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha -lactalbumin (IA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K-m for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K-m for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-TI-IA complex with UDP-Glc.Mn2+ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.