The goal of the proposed research is to elucidate fundamental physical-chemical principles that govern catalysis in enzymes. This goal will be enacted through a program of biochemical and physical studies of representatives of all three classes of coenzyme B12 (adenosylcobalamin) -dependent enzymes: Methylmalonyl-CoA mutase (MCM, Class I; human, Methylobacter extorquens), ethanolamine ammonia-lyase (EAL, Class II; Salmonella typhimurium), and Lysine-5,6-aminomutase (Class III; LAM, Clostridium stricklandii). The inquiry will be extended to address mechanism in the intracellular cobalamin (B12) trafficking pathway in humans by the CblC protein (human, Caenorhabditis elegans). Innovative methods, software and hardware for pulsed-electron paramagnetic resonance (EPR) spectroscopy, in conjunction with biochemical techniques and developed low-temperature systems enable pioneering experiments. The proposed entropically-driven, configurational catalysis mechanism will be validated and developed for the radical generation step in the Class I, II, and III B12 enzymes. The contributions of protein and coupled solvent dynamics to radical rearrangement catalysis in EAL will be augmented by high-resolution determination of the reactant and protein structures that underlie the free energy landscape. Significant biomedical and human health outcomes include: (i) Unique, fundamental knowledge about the role of multi-configurational, high-entropy protein states in enzyme function, that contribute new tools and models to the developing roadmap for leveraging these states in enzyme engineering and drug development, (ii) Characterization of EAL, which is a central player in the role of the microbiome in disease progression, including links with inflammatory bowel disease, obesity, and diabetes, (iii) Insights into the molecular mechanistic basis of human metabolic disorders identified with the cbl gene cluster, and in particular, the role of the CblC protein (also known in humans as the MMACHC, methylmalonic aciduria type C and homocystinuria, protein).
The goal of the proposed research is to elucidate fundamental physical-chemical principles that govern catalysis in enzymes. Biochemical and physical studies of representatives of all three classes of B12-dependent enzymes, and an intracellular cobalamin trafficking protein, enabled by innovations in EPR spectroscopic methods, software and hardware, will reveal how multi-configurational, high-entropy protein states contribute to enzyme function. Significant biomedical and human health outcomes include new tools and models for enzyme engineering and drug development, characterization of an enzyme that is a central player in the role of the human gut microbiome in disease progression, and insight into the molecular mechanistic basis of human metabolic disorders, including methylmalonic aciduria and homocystinuria.
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