Caccuri A M, Antonini G, Board P G, Flanagan J, Parker M W, Paolesse R, Turella P, Chelvanayagam G, Ricci G
Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy.
J Biol Chem. 2001 Feb 23;276(8):5432-7. doi: 10.1074/jbc.M002818200. Epub 2000 Oct 23.
Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.
通过稳态、预稳态动力学实验以及定点诱变技术，以硫酸1-萘酯作为共底物，对人谷胱甘肽转移酶T2-2的催化机制进行了剖析。该酶与最近进化出的属于α、π和μ类的谷胱甘肽转移酶的祖先前体相近。该酶表现出随机动力学机制，具有非常低的催化常数（k(cat)）和催化常数与米氏常数比值（k(cat)/K(m)((GSH))），且限速步骤被确定为产物释放。在稳态催化之前快速发生并导致产物积累的化学步骤，严格依赖于结合型谷胱甘肽的去质子化。用丙氨酸取代精氨酸-107会显著影响快速相，表明该残基在三元复合物中谷胱甘肽的活化和定向方面都至关重要。所有预稳态和稳态动力学数据都令人信服地符合一种动力学机制，该机制反映了这种酶相当原始的催化效率。它涉及两个缓慢相互转化或不相互转化的酶群体（或二聚体酶的活性位点），两者都能够结合并活化谷胱甘肽，且受到产物的强烈抑制。只有一个群体或亚基具有催化活性。所提出的机制解释了这种酶明显的半位点行为以及在稳态条件下观察到的明显负协同性。这些发现还暗示了谷胱甘肽转移酶家族中为优化催化活性而采用的一些进化策略，这些策略主要基于关键蛋白质片段灵活性的增加以及底物的最佳定向。
