This research program focuses on the elucidation of the enzymology of choline oxidase (CHO), an enzyme belonging to the class of flavoproteins that oxidize alcohols to aldehydes or ketones. These enzymes catalyze the transfer of a hydride equivalent from C1 of the alcohol substrate to a flavin cofactor that is tightly or covalently bound to the protein moiety. Enzyme turnover is then completed with the oxidation of the flavin via the transfer of electrons to a specific acceptor, which in CHO and other oxidases is molecular oxygen. Despite the biological importance of alcohol oxidations, the mechanisms of flavoprotein oxidases that oxidize alcohols are still not fully understood. The specific goals of this research program are to: i) Determine the timing for the proton transfers in the reduction of molecular oxygen to hydrogen peroxide. ii) Identify the catalytic base in the active site of the enzyme that initiates alcohol oxidation. iii) Establish the importance of hydrophobic residues controlling substrate access to an active site that is secluded from the bulk solvent in relation to the quantum mechanical tunneling of the hydride ion in catalysis. The experimental approaches will combine steady state kinetics, time-resolved anaerobic substrate reductions, pH and kinetic isotope effects, as well as mutagenesis and crystallography. The results that will be gathered will significantly advance the knowledge and understanding of the enzymology of CHO and of the structural and functional features that are required for efficient catalysis and oxygen reactivity in flavoprotein oxidases.
The research project will foster the integration of exciting research with the education and training of minority students, enabling them to develop skills and knowledge in mechanistic enzymology, biochemistry, protein chemistry and molecular biology. This will equip future generations of scientists with the tools that are in high-demand to fill the existing gap between the wealth of information that is available at the genomic level and the scientific knowledge at the post-genomes level, where knowledge is lagging behind. The level of training acquired by students on the project will advance the understanding of the enzyme under study. Most importantly, work on this project will provide invaluable skills and knowledge for the next generation of scientists in mechanistic enzymology . The educational activities and outreach contribution will continue and expand in the annual Southeast Enzyme Conference in Atlanta, in the Chemistry Graduate Program, and the Annual Chemistry Department Poster Day at Georgia State University.
