A histidine-based two-residue reactive site for the catalysis of hydrolysis and transesterification reactions of p-nitrophenyl esters has been engineered into a helix in a designed helix-loop-helix motif, and it has been shown to function through cooperative nucleophilic and general-acid catalysis. The two-residue site has been expanded by the incorporation of two arginine residues in the neighboring helix, and the arginines have been found to provide further transition state binding of the anionic transition state. The second-order rate constant in aqueous solution at pH 5.1 and 290 K for the peptide with the most efficient two-residue site is 0.054 M-1 s(-1), the pK(a) value of both the histidine residues, His-30 and His-34, is 5.6, and the kinetic solvent isotope effect. is 1.5. The introduction of Arg-11 and Arg-15 increases the second-order rate constant to 0.105 M-1 s(-1). The second-order rate constant of a peptide with a two-residue site of His-26 and His-30 is 0.010 M-1 s(-1), and the pK(a) values of the two His residues are 6.8 and 5.6, respectively. The difference in reactivity between the two peptides is consistent with a model where the His with the higher number in the sequence is the nucleophile and the His with the lower number is a general-acid catalyst. The results are incompatible with a model where the histidine residue with the lower number is the nucleophile.