Synthetic Interface Peptides as Inactivators of Multimeric Enzymes: Inhibitory and Conformational Properties of Three Fragments fromLactobacilluscaseiThymidylate Synthase

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Abstract
Three synthetic peptides corresponding to distinct segments of the subunit interface of the dimeric enzyme thymidylate synthase (residues 17−38, N 22; residues 174−190, M 17; and residues 201−220, C 20) have been investigated for their ability to function as inhibitors by modifying the quaternary structure of the enzyme. A dramatic reduction of enzyme activity is observed following incubation of TS with the C 20 peptide. The N 22 and M 17 peptides were unable to cause any loss of enzymatic activity. Addition of the C 20 peptide results in a loss of fluorescence of TS labeled with a dansyl group at Cys 198, following aggregation and precipitation of the protein. The effects are not observed for 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 correlate the observed function with solution structures. Peptides N 22 and M 17 are largely unstructured in aqueous solution. A population of nascent helical structures or multiple turn conformations has been detected for the C 20 peptide in aqueous solution by NMR. Addition of 50% (v/v) hexafluoroacetone trihydrate (HFA), a structure-stabilizing cosolvent, stabilizes the helical conformation in the C 20 peptide. Under similar conditions, N 22 and M 17 remain largely extended with observations of local β-turn conformations. Interestingly, the C 20 peptide is a β-hairpin in the native structure, whereas the other two peptides are individual strand components of a β-sheet.