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Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

Conway, Myra E.; Yennawar, Neela; Wallin, Reidar; Poole, Leslie B.; Hutson, Susan M.


Myra Conway
Occasional Associate Lecturer - CHSS - DAS

Neela Yennawar

Reidar Wallin

Leslie B. Poole

Susan M. Hutson


The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated β-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 Å from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 Å), requiring only a decrease of 1.1-1.5 Å. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5′-dithiobis-(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H202 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.


Conway, M. E., Yennawar, N., Wallin, R., Poole, L. B., & Hutson, S. M. (2002). Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase. Biochemistry, 41(29), 9070-9078.

Journal Article Type Article
Publication Date Jul 23, 2002
Journal Biochemistry
Print ISSN 0006-2960
Publisher American Chemical Society
Peer Reviewed Not Peer Reviewed
Volume 41
Issue 29
Pages 9070-9078
Public URL
Publisher URL