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