SYNTHETIC INTERFACE PEPTIDES AS INACTIVATORS OF MULTIMERIC ENZYMES - INHIBITORY AND CONFORMATIONAL PROPERTIES OF 3 FRAGMENTS FROM LACTOBACILLUS-CASEI THYMIDYLATE SYNTHASE

Three synthetic peptides corresponding to distinct segments of the sub unit interface of the dimeric enzyme thymidylate synthase (residues 17 -38, N 22; residues 174-190, M 17; and residues 201-220, C 20) have be en investigated for their ability to function as inhibitors by modifyi ng the quaternary structure of the enzyme. A dramatic reduction of enz yme activity is observed following incubation of TS with the C 20 pept ide. The N 22 and M 17 peptides were unable to cause any loss of enzym atic activity. Addition of the C 20 peptide results in a loss of fluor escence of TS labeled with a dansyl group at Cys 198, following aggreg ation and precipitation of the protein. The effects are not observed f or the N 22 or M 17 peptides. Loss of enzymatic activity is related to the ability of C 20 to promote protein aggregation. The conformations of the peptides have been studied using CD and NMR in order to correl ate the observed function with solution structures, Peptides N 22 and M 17 are largely unstructured in aqueous solution. A population of nas cent helical structures or multiple turn conformations has been detect ed for the C 20 peptide in aqueous solution by NMR. Addition of 50% (v /v) hexafluoroacetone trihydrate (HFA), a structure-stabilizing cosolv ent, stabilizes the helical conformation in the C 20 peptide. Under si milar conditions, N 22 and M 17 remain largely extended with observati ons of local beta-turn conformations. Interestingly, the C 20 peptide is a beta-hairpin in the native structure, whereas the other two pepti des are individual strand components of a beta-sheet.