|M.Sc Thesis||Department of Chemistry|
|Supervisor:||Prof. Keinan Ehud|
A unique structural feature that seems to dominate the 4-oxalocrotonate tautomerase (4-OT) enzyme-substrate interaction is an array of three arginine residues (Arg-61’, Arg-39” and Arg-11’) positioned in the active site. In principle, these positively charged residues might bind the negatively charged substrate via both electrostatic interactions and hydrogen bonding. Therefore, understanding the relative importance of the electrostatic and hydrogen bonding modes is of crucial importance to achieve the goal of designing new 4-OT analogs that would catalyze new reactions and exhibit specificity to new substrates.
We reasoned that replacement of these positively charged arginine residues, by nonnatural, uncharged citrulline, would eliminate electrostatic interactions with the substrate but preserve hydrogen bonding as well as the steric parameters of the active site. We also predicted that a neutral active site residue, such as the amide function of citrulline should interact more strongly with an uncharged substrate, such as amide, than with a charged one, such as a carboxylate.
This led us to the design of mutants that showed selectivity for amide substrates over the natural substrate. Furthermore, the conservative replacement of arginine by citrulline shed light on the importance of the active site electrostatics not only in binding but also in catalysis. Thus, the kinetic studies with seven new synthetic mutants, as well as with the synthetic wt-4-OT have indicated that a) the role of hydrogen bonding in substrate binding in 4-OT is at least as important as charge complimentarity, and b) one of the mutants represents a new enzyme that does not recognize the natural substrate but efficiently catalyze the tautomerization of a non-natural substrate.