Oxidation-reduction or redox cycles have been considered the exclusive domain of enzyme catalysis, but it is now clear that redox regulation, which is defined as the modulation of protein function by oxidation-reduction, is also an important signaling mechanism. We have discovered that the mammalian BCAT enzymes contain a redox-active dithiol/disulfide center. The redox-linked regulation of activity by the CXXC center demonstrates that the cysteine residues are part of a peroxide-sensing mechanism that may have an in vivo role. X-ray crystallography and site-directed mutagenesis are being used to understand the relationship between structure and function of this center in the BCAT proteins. Redox control of the BCAT proteins will be examined in cultured cells. We also have evidence that a protein web controls BCAA metabolism. We have discovered that BCATm associates with the BCKD complex and that this interaction is redox sensitive. This association suggests that BCAA catabolic enzymes can form a supramolecular complex termed a "metabolon." We are investigating the effect of redox state of BCATm on BCKD activity and phosphorylation state. The BCAT proteins will be used as "bait" to identify additional proteins that associate with human BCATc, BCATm and two alternatively spliced forms of BCATm. It has been reported that one of the BCATm isoforms is a novel co-repressor for thyroid hormone nuclear receptors. Interacting proteins will be identified by mass spectrometry. Funding NIH DK34738. Collaborators: Neela Yennawar, Pennsylvania State University (no website) and Leslie Poole and David Chuang.