Mehrotra Sonali, Balaram Hemalatha
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur Post, Bangalore 560064, India.
Biochemistry. 2007 Nov 6;46(44):12821-32. doi: 10.1021/bi701009y. Epub 2007 Oct 11.
Adenylosuccinate synthetase (AdSS) catalyzes the Mg2+ dependent condensation of a molecule of IMP with aspartate to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. AdSS from the thermophilic archaea, Methanocaldococcus jannaschii (MjAdSS) is 345 amino acids long against an average length of 430-457 amino acids for most mesophilic AdSS. This short AdSS has two large deletions that map to the middle and C-terminus of the protein. This article discusses the detailed kinetic characterization of MjAdSS. Initial velocity and product inhibition studies, carried out at 70 degrees C, suggest a rapid equilibrium random AB steady-state ordered C kinetic mechanism for the MjAdSS catalyzed reaction. AdSS are known to exhibit monomer-dimer equilibrium with the dimer being implicated in catalysis. In contrast, our studies show that MjAdSS is an equilibrium mixture of dimers and tetramers with the tetramer being the catalytically active form. The tetramer dissociates into dimers with a minor increase in ionic strength of the buffer, while the dimer is extremely stable and does not dissociate even at 1.2 M NaCl. Phosphate, a product of the reaction, was found to be a potent inhibitor of MjAdSS showing biphasic inhibition of enzyme activity. The inhibition was competitive with IMP and noncompetitive with GTP. MjAdSS, like the mouse acidic isozyme, exhibits substrate inhibition, with IMP inhibiting enzyme activity at subsaturating GTP concentrations. Regulation of enzyme activity by the glycolytic intermediate, fructose 1,6 bisphosphate, was also observed with the inhibition being competitive with IMP and noncompetitive against GTP.
腺苷酸琥珀酸合成酶(AdSS)催化一分子IMP与天冬氨酸在Mg2+依赖下缩合形成腺苷酸琥珀酸,该反应由GTP水解为GDP驱动。嗜热古菌詹氏甲烷球菌(MjAdSS)的AdSS有345个氨基酸长,而大多数嗜温AdSS的平均长度为430 - 457个氨基酸。这种短的AdSS有两个大的缺失区域,分别位于蛋白质的中部和C末端。本文讨论了MjAdSS的详细动力学特征。在70℃下进行的初速度和产物抑制研究表明,MjAdSS催化反应具有快速平衡随机AB稳态有序C动力学机制。已知AdSS表现出单体 - 二聚体平衡,二聚体参与催化作用。相比之下,我们的研究表明MjAdSS是二聚体和四聚体的平衡混合物,其中四聚体是催化活性形式。随着缓冲液离子强度略有增加,四聚体解离成二聚体,而二聚体极其稳定,即使在1.2 M NaCl浓度下也不会解离。反应产物磷酸盐被发现是MjAdSS的有效抑制剂,对酶活性表现出双相抑制作用。这种抑制作用对IMP具有竞争性,对GTP具有非竞争性。MjAdSS与小鼠酸性同工酶一样,表现出底物抑制作用,在GTP浓度未饱和时IMP会抑制酶活性。还观察到糖酵解中间产物1,6 - 二磷酸果糖对酶活性的调节作用,这种抑制作用对IMP具有竞争性,对GTP具有非竞争性。