Axarli Irene, Georgiadou Christiana, Dhavala Prathusha, Papageorgiou Anastassios C, Labrou Nikolaos E
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, GR-11855-Athens, Greece.
Biochim Biophys Acta. 2010 Apr;1804(4):662-7. doi: 10.1016/j.bbapap.2009.10.016. Epub 2009 Oct 29.
Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k(cat)) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.
植物谷胱甘肽转移酶（GSTs）在多种异源生物的代谢中起关键作用。在本报告中，通过定点诱变和稳态动力学分析研究了来自大豆（GmGSTU4-4）的tau类GSTU4-4同工酶的催化机制。在1-氯-2,4-二硝基苯（CDNB）结合反应中研究了野生型酶和三个严格保守残基（Ser13Ala、Asn48Ala和Pro49Ala）突变体的催化特性。结果表明，这些突变显著影响底物结合和特异性。Ser13Ala突变对酶催化效率的影响可以通过假设Ser13直接参与反应化学以及谷胱甘肽（GSH）和CDNB在三元催化复合物中的正确定位来解释。发现Asn48和Pro49对GSH结合位点（G位点）的结构完整性有直接作用。此外，Asn48和Pro49残基的突变可能会产生次级效应，改变酶的热稳定性和催化活性（k(cat)），而不影响催化反应限速步骤的性质。
