|Ph.D Student||Metanis Norman|
|Subject||Tailoring Enzyme Catalysis Using Synthetic Protein Chemistry|
|Department||Department of Chemistry||Supervisor||Professor Emeritus Ehud Keinan|
|Full Thesis text|
Chemical synthesis of proteins enables investigating enzyme mechanisms. We synthesized the wildtype 4-oxalocrotonate Tautomerase (4-OT), a hexameric enzyme of 62 amino acids per subunit. Seven mutants of 4-OT were synthesized by replacing the three arginine residues in the active site with the non-coded amino acid citrulline. Arg39 was found to be the most important residue for catalysis, stabilizing the negatively charged transition-state by electrostatic interactions. These findings allowed for the design and synthesis of an analogue of 4-OT that prefers a non-natural substrate to its natural substrate.
Studies with another enzyme, glutaredoxin 3 (Grx3), revealed that the replacement of cysteine by selenocysteine affected the redox potential and kinetics of this enzyme, which belongs to the thioredoxin superfamily. The preparation of enzymes containing selenocysteine is experimentally challenging. As a result, little was known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenylsulfide or diselenide bond in a protein has not been experimentally determined prior to our work.
Another study with Grx3 was based on a theoretical study, which proposed that lysine 8, a non-active site residue, interacts with the two-cysteine residues in the active site by electrostatic interactions and hydrogen bonding. Also, comparison with a related enzyme, Grx1, where this position is occupied by arginine, led to a proposal that the Arg/Lys difference is responsible for the difference in redox potential (35 mV) of these two enzymes. We replaced Lys8 in Grx3 with arginine, ornithine, citrulline and norvaline and checked both kinetics and redox potentials of these mutants. The replacements were found to lower the redox potential by 1, 4, 8 and 10 mV, respectively. The kinetic results indicated that replacing the positively charged Lys8 with norvaline, a shorter hydrophobic residue, produced a cavity that was probably filled with neutral water molecules that increased reaction rates in both directions by 10-fold.
This work also describes two new synthetic methods, which complement the original native chemical ligation (NCL) technique for the synthesis of polypeptides and proteins. One drawback of the NCL is the requirement for cysteine at the ligation site. Since cysteine is a relatively rare amino acid, there would be great advantage in a method that is based on more common amino acids. The two new methods developed here overcome this limitation, extending the ligation methodology to alanine and glycine at the ligation site.