A DESIGNED FOLDED POLYPEPTIDE MODEL SYSTEM THAT CATALYZES THE DECARBOXYLATION OF OXALOACETATE

NP-42. a 42-residue polypeptide that folds into a hairpin helix-loop-h elix motif and dimerises to form a four-helix bundle, has been designe d to catalyse the decarboxylation of oxaloacetate. The residues of the reactive site are Arg-10, Lys-11, Arg-15 and Arg-34. The lysine resid ue 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 H-1 NMR spectroscopy is 0.0 15 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 H-1 N MR spectroscopy permits the direct observation of reactants and produc ts and the problem of determining extinction coefficients is thus avoi ded. The corresponding second-order rate constant determined by UV spe ctroscopy is 0.010 M-1 s(-1). The NP-42 catalysed reaction has been sh own 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 d evelopment of efficient catalysts capable of substrate recognition.