Designed polypeptides, which fold into helix-loop-helix motifs and dimerise to four-helix bundles have been used as scaffolds for the introduction of catalytic activity for the hydrolysis of p-nitrophenyl esters. The reactive sites were based on the reactivity of histidine, and two His residues separated by four residues in the sequence was shown to be the minimal reactive site for the efficient hydrolysis of nitrophenyl esters. The efficiency increased when the HispKa values were depressed and rate enhancements of two to three orders of magnitude over that of the 4-MeIm catalysed reaction was achieved. The HisH⁺-His reactive site was versatile and catalytically competent also when the His residues were positioned three residues apart in the peptide sequence and in an interhelical configuration with one His residue in each helix. The efficiency of the reactive site was enhanced when flanked by basic residues in the opposite helix for binding of negatively charged substrates.

The reaction mechanism depended on cooperative catalysis in which nucleophilic attack by an unprotonated His residue was supplemented by general-acid catalysis by the flanking protonated His residue, by protonation of the p-nitrophenolate anion leaving group in the transition state. A kinetic solvent isotope effect was found at a pH where the nitrophenol leaving group is predominantly protonated in solution. The Hammett plot of the logarithms of the second order rate constants for ester hydrolysis versus pKa of the leaving group shows that there is approximately half a negative charge on the substrate ester oxygen in the transition state, in support of the occurrence of general acid catalysis.

At peptide concentrations below 0.1 mM some four-helix bundle motifs dissociate to form monomers and the second-order rate constants at low peptide concentrations were shown to drop due to the increased proportion of unordered structures, demonstrating a strong coupling between structure and function.

The folded polypeptide scaffold that was used initially in the design of catalytic sites, the helix-loop-helix dimer SA-42, had the properties of a molten globule in which the structure is partly unordered. The HisH⁺-His site was also introduced into helix-loop-helix dimers derived from the sequence GTD-43 which has many characteristics of a native protein and a welldefined tertiary structure. The HisH⁺-His site was shown to function also on the surface of an ordered four-helix bundle protein. However, molten globule-like structures were more efficient catalysts, especially in catalysing the hydrolysis of hydrophobic substrates, demonstrating that the surface of a molten globule is a better model system for mimicking the partially hydrophobic character of a cavity of a protein catalyst.

Cys residues were incorporated into the polypeptide sequence to form His-Cys pairs and increase the reactivity of designed polypeptide catalysts for ester hydrolysis. The Cys containing sequences were shown to react efficiently with nitrophenyl esters at a pH above 7 resulting eventually in acylation of flanking Lys residues. There was a change in mechanism below pH 5, probably reflecting a change in the nature of the nucleophilic residue. The results form the basis for the next generation of designed polypeptide catalysts for ester hydrolysis.