A designed folded polypeptide model system that catalyses the decarboxylation of oxaloacetate

Abstract

NP-42, a 42-residue polypeptide that folds into a hairpin helix-loop-helix motif and dimerises to form a four-helix bundle, has been designed to catalyse the decarboxylation of oxaloacetate. The residues of the reactive site are Arg-10, Lys-11, Arg-15 and Arg-34. The lysine residue reacts with the carbonyl carbon of the substrate to form the imine intermediate that is decarboxylated to form the pyruvate product. The second-order rate constant of the NP-42 catalysed reaction in aqueous solution at pH 7.0 and 298 K determined by ¹H NMR spectroscopy is 0.015 M^–1 s^–1, which is approximately a factor of 10 larger than that of the butylamine catalysed reaction. The study of the reaction by ¹H NMR spectroscopy permits the direct observation of reactants and products and the problem of determining extinction coefficients is thus avoided. The corresponding second-order rate constant determined by UV spectroscopy is 0.010 M^–1 s^–1. The NP-42 catalysed reaction has been shown not to follow saturation kinetics and the reaction follows pseudo-first-order kinetics over a range of substrate concentrations from 2.5 to 30 mM. NP-42 is thus a well-defined model system for the further development of efficient catalysts capable of substrate recognition.

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