|Ph.D Student||Sluszny Chanan|
|Subject||Analysis of Polycyclic Aromatic Hydrocarbons in|
|Department||Department of Chemistry||Supervisor||Professor Israel Schechter|
Part 1: Design of new enzymes that operate via Schiff base intermediates.
A profound change in the catalytic activity and mechanism of 4-oxalocrotonate tautomerase (4-OT) was accomplished by a rationally designed single amino acid substitution that corresponds to a single base pair mutation. The wild-type enzyme catalyzes only the tautomerization of oxalocrotonate, while the P1A mutant catalyzes two reactions - the original tautomerization reaction and the decarboxylation of oxaloacetate. Although the N-terminal amine of P1A is involved in both reactions, our results support a nucleophilic mechanism for the decarboxylase activity in contrast to the general acid/base mechanism that has been established for the tautomerase activity.
Part 2. The product of the natural reaction catalyzed by 4-oxalocrotonate tautomerase becomes an affinity label of its mutant.
Analysis of P1A and P1G mutants by mass spectrometry demonstrated that P1A is susceptible to a 1,4-addition of the N-terminal primary amine across the double bond of enone 3 to form a covalent adduct. Although slower than the isomerization reaction, the addition is fast, with 50% of the active sites being alkylated within 12 min. By contrast, the wt4-OT shows no detectable modification over 24 hours.
Part 3. Protein Synthesis by Solid Phase Chemical Ligation.
A new strategy was developed for the assembly of large polypeptides on a solid support that utilizes a highly stable safety catch acid labile (SCAL) linker. The utility of the methodology is demonstrated by a three segment synthesis of vMIP I, a chemokine that contains all twenty natural amino acids and has two disulfide bonds.