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Inactivation of Glyceraldehyde-3-Phosphate Dehydrogenase by Fumarate in Diabetes: Formation of S-(2-succinyl)cysteine, a Novel Chemical Modification of Protein and Possible Biomarker of Mitochondrial Stress

¹The Department of Chemistry and Biochemistry, University of South Carolina

Objective: S-(2-succinyl)cysteine (2SC) is formed by a Michael addition reaction between the Krebs cycle intermediate, fumarate, and cysteine residues in protein. We have investigated the role of fumarate in chemical modification and inhibition of the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in vitro and in tissues of diabetic rats.

Research Design and Methods: GAPDH was incubated with fumarate in phosphate buffer to assess effects of fumarate on enzyme activity in vitro. Sites of 2SC formation were determined by analysis of tryptic peptides by HPLC-quadrupole-time of flight (QTOF) mass spectrometry (MS). 2SC and fumarate in gastrocnemius muscle of control and streptozotocin-induced diabetic rats were measured by LC/MS/MS and GC/MS, respectively. GAPDH was isolated from muscle by immunoprecipitation, and sites of modification of GAPDH were determined by MS analysis.

Results: 2SC was found, both in vitro and in vivo, about equally at active site Cys-149 and nucleophilic Cys-244. Inactivation of GAPDH by fumarate in vitro correlated with formation of 2SC. In diabetic compared to control rats, fumarate and 2SC concentration increased 5-fold, accompanied by an 25% decrease in GAPDH specific activity. The fractional modification of GAPDH by 2SC was significantly increased in diabetic vs. control animals, consistent with the decreased specific activity of GAPDH in muscle of diabetic animals.

Conclusion: Fumarate contributes to inactivation of GAPDH in diabetes. 2SC may be a useful biomarker of mitochondrial stress in diabetes. Modification of GAPDH and other enzymes and proteins by fumarate may contribute to the metabolic changes underlying the development of diabetic complications.

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