Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation
Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation
复制标题
通过 C 末端截短减少嗜热脂肪地芽孢杆菌苹果酸脱氢酶的底物抑制
DOI：
10.1093/protein/gzac008
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Nishiya Yoshiaki
中科院分区：
文献类型：
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作者：
Shimozawa Yuya;Matsuhisa Hinano;Nakamura Tsutomu;Himiyama Tomoki;Nishiya Yoshiaki
苹果酸脱氢酶（MDH）催化草酰乙酸还原为l -苹果酸。使用嗜热硬脂地杆菌mdh （gs-MDH）作为诊断试剂；然而，gs-MDH在高底物浓度下被强烈抑制，这限制了其反应速率。在这里，我们通过删除gs-MDH的c端残基来降低底物对其的抑制作用。计算分析表明，c端残基调节了活性位点环的位置。gs-MDH的c端缺失成功地将k值提高了5- 8倍，并保持了热稳定性（约为野生型酶的90%），尽管cat/ km值降低了不到2倍。突变体的结构显示出活性位点环的位置发生了变化，其体积减小，这表明通过消除引起抑制的假定底物结合位点，底物抑制减少了。我们的研究结果提供了一种有效的方法来减少底物对酶的抑制，而不损失其他参数，包括结合常数和稳定性常数。
Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate.Geobacillus stearothermophilusMDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed that C-terminal residues regulate the position of the active site loop. C-terminal deletions of gs-MDH successfully increasedKivalues by 5- to 8-fold with maintained thermal stability (>90% of the wild-type enzyme), althoughkcat/Kmvalues were decreased by <2-fold. The structure of the mutant showed a shift in the location of the active site loop and a decrease in its volume, suggesting that substrate inhibition was reduced by eliminating the putative substrate binding site causing inhibition. Our results provide an effective method to reduce substrate inhibition of the enzyme without loss of other parameters, including binding and stability constants.