KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix-loop-helix motif that dimerizes and forms a four-helix bundle. The solution structure of the folded KO-42 dimer has been determined by NMR and CD spectroscopy and ultracentrifugation. On the surface of the folded polypeptide a reactive site has been engineered that is capable of catalyzing acyl-transfer reactions of reactive esters, The reactive site of KO-42 contains six histidine residues with perturbed pK(a) values. The pK(a)s of His-15, His-30, and His-34 are close to 5, whereas those of His-11, His-19, and His-26 are close to 7, with nonideal titration curves. The second-order rate constant for the KO-42 catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 4.1 and 290 K is 0.1 M-1 s(-1), which is 1140 times larger than that of the 4-methylimidazole (4-MeIm) catalyzed reaction, 8.8 x 10(-5) M-1 s(-1). The second-order rate constant for the KO-42 catalyzed transesterification of mono-p-nitrophenyl fumarate to form the corresponding trifluoroethyl ester in 10 vol % trifluoroethanol at pH 4.1 and 290 K is 0.052 M-1 s(-1) which is 620 times larger than that of the 4-MeIm catalyzed reaction, 8.4 x 10(-5) M-1 s(-1). KO-42 catalyzes the corresponding reactions of other p-nitrophenyl esters with similar rate enhancements. At pH 4.1 in aqueous solution where the rate constant ratio k(2)(KO-42)/k(2)(4-MeIm) is larger than 10(3) the predominant reactive species of KO-42 have unprotonated histidines flanked by protonated histidines. The kinetic solvent isotope effect at pH 4.7 is 2.0 which shows that isotopic fractionation occurs in the transition state. The kinetic solvent isotope effect at pH 6.1 is 1.1 which shows that there is neither general acid-general base catalysis nor strong hydrogen bonding in the transition state of the rate-limiting reaction step at that pH. The results suggest that at low pH the dominant catalytic species functions through a mechanism where unprotonated nucleophilic histidines are flanked by protonated histidines that bind to one or both of the ester oxygens in the transition state.