Identification of lysine 153 as a functionally important residue in UDP-galactose 4-epimerase from Escherichia coli.
Identification of lysine 153 as a functionally important residue in UDP-galactose 4-epimerase from Escherichia coli.
复制标题
鉴定赖氨酸 153 作为大肠杆菌 UDP-半乳糖 4-差向异构酶中功能重要的残基。
DOI：
10.1021/bi00211a035
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Frey,PA
中科院分区：
文献类型：
--
作者：
Swanson,BA;Frey,PA
1993年9月22日收到的修订版Mandalpt ®摘要：通过对突变酶的定点诱变和动力学及分光光度分析，研究了赖氨酸153在大肠杆菌UDP-半乳糖4-差向异构酶作用中的作用。UDP-半乳糖4-差向异构酶的晶体结构表明，NAD+与辅酶位点的结合包括赖氨酸153的e-铵基团与烟酰胺核苷的2 ′-和3 ′-羟基的氢键相互作用。这个残基突变为甲硫氨酸或丙氨酸会使酶的催化活性降低103倍以上。与野生型酶相关的NAD”* 1”受到糖如葡萄糖和阿拉伯糖的UMP依赖性还原，但突变蛋白K153 M和K153 A在存在或不存在UMP的情况下不被糖还原。与野生型酶相关的NAD”1”也受到氰基硼氢化钠的UMP依赖性还原。然而，尽管突变蛋白与UMP结合得很好，但与它们相关的NAD+被氰基硼氢化钠还原的速率对UMP的存在几乎不敏感。纯化的野生型酶含有显著量的结合到辅酶位点的NADH;然而，纯化的突变体K153 M和K153 A含有非常少的NADH。我们的结论是，赖氨酸153在增加酶结合的NAD”1”的化学反应性中起着重要作用，尿苷核苷酸依赖的构象变化与还原失活和UDP-半乳糖4-差向异构酶的催化活性相关。
Revised Manuscript Received September 22, 1993® abstract: The role of lysine 153 in the action of UDP-galactose 4-epimerase from Escherichia coli has been investigated by site specific mutagenesis and kinetic and spectrophotometric analysis of the mutant enzymes. Thecrystal structure of UDP-galactose 4-epimerase shows that the binding of NAD+ to the coenzyme site includes the hydrogen bonded interaction of the e-ammonium group of lysine 153 with the 2'-and 3'-hydroxyl groups of the nicotinamide riboside. Mutationof this residue to methionine or alanine decreases the catalytic activity of the enzyme by á factor of more than 103. The NAD"* 1" associated with the wild type enzyme is subject to UMP-dependent reduction bysugars such as glucose and arabinose, but the mutant proteins K153M and K153A are not reduced by sugars in thepresence or absence of UMP. NAD" 1" associated with the wild type enzyme is also subject to UMP-dependent reduction by sodium cyanoborohydride. However, although the mutant proteins bind UMP very well, the rate at which NAD+ associated with them is reduced by sodium cyanoborohydride is almost insensitive to the presence of UMP. The purified wild type enzyme contains significant amounts of NADH bound to the coenzyme site; however, the purified mutants K153M and K153A contain very little NADH. We conclude that lysine 153 plays an important role in increasing the chemical reactivity of enzyme-bound NAD" 1" in the uridine nucleotidedependent conformational change associated with reductive inactivation and the catalytic activityof UDP-galactose 4-epimerase.