Mutation of arginine 228 to lysine enhances the glucosyltransferase activity of bovine beta-1,4-galactosyltransferase I.
Mutation of arginine 228 to lysine enhances the glucosyltransferase activity of bovine beta-1,4-galactosyltransferase I.
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精氨酸 228 突变为赖氨酸可增强牛 β-1,4-半乳糖基转移酶 I 的葡糖基转移酶活性。
DOI：
10.1021/bi0479454
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Qasba,PradmanK
中科院分区：
文献类型：
--
作者：
Ramakrishnan,Boopathy;Boeggeman,Elizabeth;Qasba,PradmanK
β-1，4-半乳糖基转移酶I（β4Gal-T1）通常在Mn 2+离子存在下将Gal从UDP-Gal转移至GlcNAc（Gal-T活性），也将Glc从UDP-Glc转移至GlcNAc（Glc-T活性），尽管效率仅为0.3%。此外，α-乳白蛋白（LA）可使Glc-T活性增强25倍以上。UDP-Gal-和UDP-Glc-结合的β4Gal-T1的晶体结构的比较揭示了Gal和Glc部分中的O 4羟基与Glu 317的侧链羧酸酯基团形成氢键。葡萄糖的O 4羟基的取向导致Glu 317的侧链羧酸基团的空间位阻，解释了酶的低Glc-T活性。在这项研究中，我们表明，Arg 228，在Glu 317附近的一个残基，赖氨酸（R228 K-Gal-T1）的突变，结果在一个高15倍的Glc-T活性，这是进一步增强LA的近25%的野生型的Gal-T活性。动力学参数表明，Arg 228突变为赖氨酸的主要影响是Glc-T的kcat，在不存在和存在LA的情况下，其增加了3 - 4倍;同时，Gal-T反应的kcat降低了30倍。与LA、UDP-Gal和Mn 2+复合的R228 K-Gal-T1的晶体结构在1.9 μ m分辨率下测定，表明与野生型相比，Asp 318侧链表现出较小的交替构象。这种构象的改变对UDP-Gal的Gal部分的O 4羟基产生了空间位阻，可能导致UDP-Gal的解离和Gal-T反应的kcat降低。
β-1,4-Galactosyltransferase I (β4Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn2+ion (Gal-T activity) and also transfers Glc from UDP-Glc to GlcNAc (Glc-T activity), albeit at only 0.3% efficiency. In addition, α-lactalbumin (LA) enhances this Glc-T activity more than 25 times. Comparison of the crystal structures of UDP-Gal- and UDP-Glc-bound β4Gal-T1 reveals that the O4 hydroxyl group in both Gal and Glc moieties forms a hydrogen bond with the side chain carboxylate group of Glu317. The orientation of the O4 hydroxyl of glucose causes a steric hindrance to the side chain carboxylate group of Glu317, accounting for the enzyme's low Glc-T activity. In this study, we show that mutation of Arg228, a residue in the vicinity of Glu317, to lysine (R228K-Gal-T1) results in a 15-fold higher Glc-T activity, which is further enhanced by LA to nearly 25% of the Gal-T activity of the wild type. The kinetic parameters indicate that the main effect of the mutation of Arg228 to lysine is on thekcatof Glc-T, which increases 3−4-fold, both in the absence and in the presence of LA; simultaneously, thekcatfor the Gal-T reaction is reduced 30-fold. The crystal structure of R228K-Gal-T1 complexed with LA, UDP-Gal, and Mn2+determined at 1.9 Å resolution shows that the Asp318 side chain exhibits a minor alternate conformation, compared to that in the wild type. This alternate conformation now causes a steric hindrance to the O4 hydroxyl group of the Gal moiety of UDP-Gal, probably causing the dissociation of UDP-Gal and the reducedkcatof the Gal-T reaction.