Aceto A, Dragani B, Allocati N, Masulli M, Petruzzelli R, Di Ilio C
Istituti di Scienze Biochimiche e Medicina Sperimentale, Facoltà di Medicina, Università G. D'Annunzio, Chieti, Italy.
Arch Biochem Biophys. 1996 Apr 15;328(2):302-8. doi: 10.1006/abbi.1996.0177.
The proteolytic attack of bacterial glutathione S-transferase (GSTB1-1) by trypsin cleaves and inactivates the enzyme. The polypeptide portion of GSTB1-1 encompassing the cleavage site (Lys35-Lys36) constitutes an exposed and flexible region of the GSTB1-1 G-site. By sequentially using a benzamidine-affinity chromatography and GSH-affinity column, a proteolyzed form of GSTB1-1 (23/20 kDa), in which only one subunit has been cleaved has been purified and characterized. Gel filtration, sequence analysis of subunits separated by HPLC, and CD experiments indicate that the 23/20-kDa GSTB1-1 form is a dimer and maintains its secondary structure. In addition, kinetic determinations reveal that the proteolytic cleavage of one polypeptide chain inactivates one active site but does not influence the catalytic efficiency of the second one. Previous refolding studies on GSTB1-1 have shown that the formation of a correct dimer precedes the recovery of the full activity of the enzyme, indicating that the dimeric structure is essential for catalytic activity of GSTB1-1. Thus, although GSTB1-1 active sites are catalytically independent and, probably, mainly located on each monomer, interactions deriving from the dimeric arrangement of the molecule appear essential for maintaining each active site in a fully active conformation. The catalytic independence of the two active sites, as well as the importance of dimeric structure for catalytic activity, has already been established for other GSTs. Thus, despite the very low sequence identity and kinetic differences between bacterial and other distant members of the GST superfamily, the results reported here indicate that important properties of the GST active site are conserved.
胰蛋白酶对细菌谷胱甘肽S-转移酶（GSTB1-1）的蛋白水解攻击会使该酶裂解并失活。GSTB1-1包含裂解位点（Lys35-Lys36）的多肽部分构成了GSTB1-1 G位点一个暴露且灵活的区域。通过依次使用苯甲脒亲和色谱法和谷胱甘肽亲和柱，已纯化并鉴定出一种仅一个亚基被裂解的GSTB1-1蛋白水解形式（23/20 kDa）。凝胶过滤、通过高效液相色谱法分离的亚基的序列分析以及圆二色实验表明，23/20-kDa的GSTB1-1形式是二聚体并维持其二级结构。此外，动力学测定表明，一条多肽链的蛋白水解裂解会使一个活性位点失活，但不会影响另一个活性位点的催化效率。先前对GSTB1-1的复性研究表明，正确二聚体的形成先于该酶完全活性的恢复，这表明二聚体结构对于GSTB1-1的催化活性至关重要。因此，尽管GSTB1-1活性位点在催化上是独立的，并且可能主要位于每个单体上，但分子二聚体排列产生的相互作用似乎对于将每个活性位点维持在完全活性构象中至关重要。对于其他谷胱甘肽S-转移酶，两个活性位点的催化独立性以及二聚体结构对催化活性的重要性已经得到证实。因此，尽管细菌谷胱甘肽S-转移酶超家族与其他远缘成员之间的序列同一性非常低且动力学存在差异，但此处报道的结果表明谷胱甘肽S-转移酶活性位点的重要特性是保守的。
