TY - JOUR
T1 - The interactions of 9,10-phenanthrenequinone with glyceraldehyde-3- phosphate dehydrogenase (GAPDH), a potential site for toxic actions
AU - Rodriguez, Chester E.
AU - Fukuto, Jon M.
AU - Taguchi, Keiko
AU - Froines, John
AU - Cho, Arthur K.
N1 - Funding Information:
The research described in this article has been funded by the United States Environmental Protection Agency through Grant #CR-82805901 to UCLA. It has not been subjected to the Agency's required peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred.
PY - 2005/6/30
Y1 - 2005/6/30
N2 - 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 (Ki) 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.
AB - 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 (Ki) 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.
KW - 1,4-Benzoquinone
KW - 9,10-Phenanthrenequinone
KW - GAPDH
KW - Hydrogen peroxide
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U2 - 10.1016/j.cbi.2005.05.002
DO - 10.1016/j.cbi.2005.05.002
M3 - Article
C2 - 15950210
AN - SCOPUS:20644457548
VL - 155
SP - 97
EP - 110
JO - Chemico-Biological Interactions
JF - Chemico-Biological Interactions
SN - 0009-2797
IS - 1-2
ER -