Mechanistic studies on CDP-6-deoxy-delta 3,4-glucoseen reductase: the role of cysteine residues in catalysis as probed by chemical modification and site-directed mutagenesis.
Mechanistic studies on CDP-6-deoxy-delta 3,4-glucoseen reductase: the role of cysteine residues in catalysis as probed by chemical modification and site-directed mutagenesis.
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CDP-6-脱氧-δ 3,4-葡萄糖烯还原酶的机理研究：通过化学修饰和定点诱变探讨半胱氨酸残基在催化中的作用。
DOI：
10.1021/bi00013a003
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Liu,HW
中科院分区：
文献类型：
--
作者：
Ploux,O;Lei,Y;Vatanen,K;Liu,HW
修订版Mandarin pt接收日期：1994年11月14日®摘要：CDP-6-deoxy-A3| 4-葡萄糖还原酶（E3）在大肠杆菌中得到了高效表达（比野生型菌株高670倍），该酶在假结核耶尔森氏菌脂多糖中发现了一种3，6-二脱氧己糖--CDP-蛔虫糖的形成过程中催化C-3脱氧步骤的还原。该黄素酶也含有一个植物铁氧还蛋白类型[2Fe-2S]簇，其被5，5 '-二硫代双（2-硝基苯甲酸）（DTNB）和N-乙基马来酰亚胺灭活。在这两种情况下，失活遵循伪一级动力学。DTNB与E3反应的二级速率常数为0.25 mM-1 min-1（20 ℃，pH8.0）.失活酶的详细表征表明，无论是黄素，也不是[2Fe-2S]簇在失活过程中被改变。由于这种失活是可逆的处理失活的酶与1 mM的D，L-二硫苏糖醇（DTT），它的结论是，只有半胱氨酸残基在失活过程中被修改。使用Tsou开发的方法分析失活显示，两个半胱氨酸以相似的速率与DTNB反应，并且任何一个的修饰都足以削弱E3的活性。用N-乙基[2，3- 14 C]马来酰亚胺标记的E3的胰蛋白酶消化，然后通过高效液相色谱法分级分离消化物，得到两种标记的肽，这两种肽都作为一对可相互转化的非对映异构体单独分离。这些标记的肽的序列分析允许鉴定Cys-75和Cys-296作为反应性半胱氨酸残基。有趣的是，C75 S和C296 S突变体蛋白表现出与野生型酶相同的物理性质和相当的催化性质。由于Cys-296是属于同一类别的酶的NAD（P）结合结构域中的保守残基，因此该残基可能参与稳定E3和NADH之间的电荷转移复合物，从而促进烟酰胺核苷酸向黄素的电荷转移。化学修饰的Cys-75紧邻E3中的[2 Fe-2S]中心，可能会阻止氧化还原中心的适当并置，从而阻碍电子转移，导致酶失活。这些结果可能是有用的放置上的肽折叠的限制，包括活性位点的E3之间的电子转移的NADH，FAD，和[2Fe-2S]中心。
Revised Manuscript Received November 14, 1994® abstract: CDP-6-deoxy-A3| 4-glucoseen reductase (E3), which catalyzes the reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3, 6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis, has been expressed at high level in Escherichia coli (670 times over the wild-type strain). This flavoenzyme, which also contains one plant ferredoxin type [2Fe-2S] cluster, was inactivated by 5, 5'-dithiobis (2-nitrobenzoic acid)(DTNB) and/V-ethylmaleimide. In both cases the inactivation followed a pseudo first order kinetics. The second order rate constant for the reaction of DTNB with E3 was 0.25 mM-1 min-1 at 20 C, pH 8.0. Detailed characterization of the inactivated enzyme showed that neither the flavin nor the [2Fe-2S] cluster was altered during inactivation. Since this inactivation was reversible by treating the inactivated enzyme with 1 mM D, L-dithiothreitol (DTT), it was concluded that only cysteine residues were modified during inactivation. Analysis of the inactivation using the method developed by Tsou revealed that two cysteines react with DTNB at similar rates and modification of either one is enough to impair E3’s activity. Tryptic digestion of E3 labeled with/V-ethyl [2, 3-14C] maleimide, followed by fractionation of the digest by high performance liquid chromatography, gave two labeled peptides, both of which were separately isolated as a pair of interconvertible diastereoisomers. Sequence analysis of these labeled peptides allowed the identification of Cys-75 and Cys-296 as the reactive cysteine residues. Interestingly, the C75S and C296S mutant proteins exhibit identical physical and comparable catalytic properties as the wild-type enzyme. Since Cys-296 is a conserved residue in the NAD (P) bindingdomain of enzymes belonging to the same class, this residue may be involved in stabilizing the charge-transfer complex between E3 and NADH, thus facilitating hydridetransfer from the nicotinamide nucleotide to flavin. A chemically modified Cys-75 which is immediately adjacentto the [2Fe-2S] center in E3 may prevent the properjuxtaposition of the redox centers and thus impede electron transfer leadingto enzyme inactivation. These results may be useful for placing constraints on the peptide folding comprising the active site of E3 for electron transfer between NADH, FAD, and the [2Fe-2S] center.