RESUMO
An 18-residue miniature enzyme, Apoxaldie-1, has been designed, based on the known structure of the neurotoxic peptide apamin. Three lysine residues were introduced on the solvent-exposed face of the apamin alpha-helix to serve as an active site for decarboxylation of oxaloacetate. The oxidised form of Apoxaldie-1, in which two disulfide bonds stabilise the alpha-helix, formed spontaneously. CD spectroscopy measurements revealed that, in its oxidised form, Apoxaldie-1 adopted a stably folded structure, which was lost upon reduction of the disulfide bonds. Despite its small size and the absence of a designed binding pocket, Apoxaldie-1 displayed saturation kinetics in its oxidised form and catalysed the decarboxylation of oxaloacetate at a rate that was almost four orders of magnitude faster than that observed with n-butylamine. This rivals the performance of the best synthetic oxaloacetate decarboxylases reported to date. Unlike those, however, Apoxaldie-1 displayed significant stability. It maintained its secondary structure at temperatures in excess of 75 degrees C, in the presence of high concentrations of guanidinium chloride and at pH values as low as 2.2. Apamin-based catalysts have potential for the generation of miniature peptides that display activity under nonphysiological conditions.
