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首页> 外文期刊>Biochemistry >Probing catalysis by escherichia coli dTDP-glucose-4,6-dehydratase:identification and preliminary characterization of functioal amino acid residues at the active site
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Probing catalysis by escherichia coli dTDP-glucose-4,6-dehydratase:identification and preliminary characterization of functioal amino acid residues at the active site

机译：大肠杆菌dTDP-葡萄糖-4,6-脱水酶的催化催化作用：活性位点上功能性氨基酸残基的鉴定和初步表征

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A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase. The active site configuration is consistent with the partially refined 3-dimensional structure of 4,6-dehydratase, which lacks substrate—nucleotide but contains NAD~ (PDB file lBXK). From the model, two groups of active site residues were identified. The first group consists of Aspl35DEH, Glu136DEH, Glu198DEH, Lys199DEH, and Tyr3O1DEH. These residues are near the substrate— pyranose binding pocket in the model, they are completely conserved in 4,6-dehydratase, and they differ from the corresponding equally well-conserved residues in 4-epimerase. The second group of residues is Cys187DEH, Asn19ODEH, and His232DEH, which form a motif on the re face of the cofactor nicotinamide binding pocket that resembles the catalytic triad of cysteine-proteases. The importance of both groups of residues was tested by mutagenesis and steady-state kinetic analysis. In all but one case, a decrease in catalytic efficiency of approximately 2 orders of magnitude below wild-type activity was observed. Mutagenesis of each of these residues, with the exception of Cys 1 87DEH, which showed near-wild-type activity, clearly has important negative consequences for catalysis. The allocation of specific functions to these residues and the absolute magnitude of these effects are obscured by the complex chemistry in this multistep mechanism. Tools will be needed to characterize each chemical step individually in order to assign loss of catalytic efficiency to specific residue functions. To this end, the effects of each of these variants on the initial dehydrogenation step were evaluated using a the substrate analogue dTDP-xylose. Additional steady-state techniques were employed in an attempt to further limit the assignment of rate limitation. The results are discussed within the context of the 4,6-dehydratase active site model and chemical mechanism.
机译：通过将氨基酸序列比对信息与UDP-半乳糖-4-表异构酶的3维结构相结合，已生成了大肠杆菌dTDP-葡萄糖-4,6-脱水酶（4,6-脱水酶）活性位点的模型。活性位点配置与4,6-脱水酶的部分精制3维结构一致，该结构缺少底物核苷酸，但含有NAD〜（PDB文件lBXK）。从模型中鉴定出两组活性位点残基。第一组由Aspl35DEH，Glu136DEH，Glu198DEH，Lys199DEH和Tyr3O1DEH组成。这些残基在模型中的底物-吡喃糖结合口袋附近，在4,6-脱水酶中完全保守，并且与在4-表异构酶中相应的同样保守的残基不同。第二组残基是Cys187DEH，Asn19ODEH和His232DEH，它们在辅助因子烟酰胺结合口袋的背面形成了一个基序，类似于半胱氨酸蛋白酶的催化三联体。通过诱变和稳态动力学分析测试了两组残基的重要性。除一种情况外，在所有情况下，均观察到催化效率比野生型活性低约2个数量级。除Cys 1 87DEH表现出近乎野生型的活性外，所有这些残基的诱变作用显然对催化具有重要的负面影响。在这些多步机理中，复杂的化学反应掩盖了对这些残基的特定功能分配以及这些效应的绝对强度。将需要工具来分别表征每个化学步骤，以便将催化效率的损失分配给特定的残留功能。为此，使用底物类似物dTDP-木糖评估了这些变体对初始脱氢步骤的影响。为了尝试进一步限制速率限制的分配，采用了其他稳态技术。在4,6-脱水酶活性位点模型和化学机理的背景下讨论了结果。

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