Polypeptides with supersecondary structures as templates in rational catalyst design. Catalysis of self functionalization by designed helix–loop–helix motifs

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Abstract
Three designed polypeptides with 42 amino acids each, SA-42, RA-42 and PA-42, were engineered to catalyse acyl-transfer reactions of mono-p-nitrophenylfumarate I. The second-order rate constants of the peptides for the formation of p-nitrophenol were larger than that of the background reaction with factors of 331, 1750 and 1937, respectively. The background reaction is the reaction between I and trifluoroethoxide ion to form the trifluoroethyl ester. The second-order rate constant for the 4-methylimidazole catalysed reaction between I and trifluoroethoxide ion was 211 times larger than that of the background reaction and the similarity in rate constants suggests that the histidine residues in the polypeptides act as nucleophilic catalysts. The observed rate increases in RA-42 and PA-42 over that of SA-42 identifies the location of the reaction centre. The identity of the reaction products in the RA-42 catalysed reaction was established by NMR spectroscopy and mass spectrometry. The side chain of ornithine-15 was acylated by the fumaryl residue. Thus, in a fast and selective second reaction step the fumaryl group was transferred from histidine-11 to the side chain of ornithine-15. The ability of a designed helix–loop–helix motif, RA-42, to catalyse its own functionalization is thereby demonstrated for the first time.