Rodriguez Chester E, Fukuto Jon M, Taguchi Keiko, Froines John, Cho Arthur K
Department of Pharmacology, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, CA 90095-1735, USA.
Chem Biol Interact. 2005 Jun 30;155(1-2):97-110. doi: 10.1016/j.cbi.2005.05.002.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate, one of the precursors for glycolytic ATP biosynthesis. The enzyme contains an active site cysteine thiolate, which is critical for its catalytic function. As part of a continuing study of the interactions of quinones with biological systems, we have examined the susceptibility of GAPDH to inactivation by 9,10-phenanthrenequinone (9,10-PQ). In a previous study of quinone toxicity, this quinone, whose actions have been exclusively attributed to reactive oxygen species (ROS) generation, caused a reduction in the glycolytic activity of GAPDH under aerobic and anaerobic conditions, indicating indirect and possible direct actions on this enzyme. In this study, the effects of 9,10-PQ on GAPDH were examined in detail under aerobic and anaerobic conditions so that the role of oxygen could be distinguished from the direct effects of the quinone. The results indicate that, in the presence of the reducing agent DTT, GAPDH inhibition by 9,10-PQ under aerobic conditions was mostly indirect and comparable to the direct actions of exogenously-added H2O2 on this enzyme. GAPDH was also inhibited by 9,10-PQ anaerobically, but in a somewhat more complex manner. This quinone, which is not considered an electrophile, inhibited GAPDH in a time-dependent manner, consistent with irreversible modification and comparable to the electrophilic actions of 1,4-benzoquinone (1,4-BQ). Analysis of the anaerobic inactivation kinetics for the two quinones revealed comparable inactivation rate constants (k(inac)), but a much lower inhibitor binding constant (K(i)) for 1,4-BQ. Protection and thiol titration studies suggest that these quinones bind to the NAD+ binding site and modify the catalytic thiol from this site. Thus, 9,10-PQ inhibits GAPDH by two distinct mechanisms: through ROS generation that results in the oxidization of GAPDH thiols, and by an oxygen-independent mechanism that results in the modification of GAPDH catalytic thiols.
甘油醛-3-磷酸脱氢酶（GAPDH）催化甘油醛3-磷酸氧化磷酸化为1,3-二磷酸甘油酸，这是糖酵解ATP生物合成的前体之一。该酶含有一个活性位点半胱氨酸硫醇盐，这对其催化功能至关重要。作为醌与生物系统相互作用持续研究的一部分，我们研究了GAPDH对9,10-菲醌（9,10-PQ）失活的敏感性。在先前关于醌毒性的研究中，这种醌的作用一直完全归因于活性氧（ROS）的产生，在有氧和无氧条件下均导致GAPDH的糖酵解活性降低，表明对该酶有间接和可能的直接作用。在本研究中，在有氧和无氧条件下详细研究了9,10-PQ对GAPDH的影响，以便区分氧的作用与醌的直接作用。结果表明，在存在还原剂二硫苏糖醇（DTT）的情况下，9,10-PQ在有氧条件下对GAPDH的抑制主要是间接的，并且与外源添加的过氧化氢（H2O2）对该酶的直接作用相当。GAPDH在无氧条件下也被9,10-PQ抑制，但方式更为复杂。这种醌不被认为是亲电试剂，它以时间依赖性方式抑制GAPDH，这与不可逆修饰一致，并且与1,4-苯醌（1,4-BQ）亲电作用相当。对这两种醌的无氧失活动力学分析显示失活速率常数（k(inac)）相当，但1,4-BQ的抑制剂结合常数（K(i)）要低得多。保护和硫醇滴定研究表明，这些醌与NAD+结合位点结合并修饰该位点的催化硫醇。因此，9,10-PQ通过两种不同机制抑制GAPDH：通过产生ROS导致GAPDH硫醇氧化，以及通过不依赖氧的机制导致GAPDH催化硫醇修饰。
