This collaborative project involves a combined theoretical-experimental approach that aims at a better understanding of proton transfer processes in enzymes. A specific focus will be placed on the carbonic anhydrase family, in which intramolecular proton transfer is the rate-limiting step. To guide the experimental design of new and more efficient proton transfer pathways in carbonic anhydrase, a series of calculations will be carried out using QM/MM methods based on a recently proposed approximate density functional theory (SCC-DFTB) and the CHARMM force field. These calculations will provide microscopic information about the free energy barrier, rate constant, solvent effect, kinetic isotope effect, and more importantly the contribution of individual residues to the proton transfer process. On the experimental side, site-directed mutagenesis of selected active site residues of the enzyme will be carried out to introduce chemically modified amino acids containing artificial proton shuttles; the location and nature of these groups will be guided by computational studies. Structural measurements of modified enzymes using x-ray crystallography will yield valuable information about the spatial organizations of the active site, such as the orientation of the new shuttle group and its distance to the Zn ion as well as the configuration of water bridges between the proton donor and acceptor groups. Kinetic studies, utilizing isotopic substitutions and pH changes, will reveal the catalytic effectiveness of the mutation(s). The strength of the present project lies in a coherent interplay between state-of-the-art experimental and theoretical investigations. Experimental results help to define the key questions for theoretical simulations, while in turn theoretical studies provide both interpretation of experimental findings and guidance to further experimental exploration.
Broader Impact: In addition to providing mechanistic insights into the function of carbonic anhydrase, the present project has broader impacts on the understanding of proton transfers in other enzyme systems and on uncovering general principles for de novo protein engineering. The research will also provide ample opportunities for participating students to acquire important skills in computational chemistry, organic and biological chemistry, molecular biology, and structural biology. Finally, the integrated theoretical-experimental research will be invaluable for motivating undergraduate students and attracting them to scientific research and discovery.
