Versatility of helix–loop–helix catalysts — cooperative HisH+–His sites that span both helices

Abstract

Peptides with 42 amino acid residues have been designed to fold into helix-loop-helix motifs that dimerize to form four-helix bundles and catalyze the hydrolysis of p-nitrophenyl esters. Their reactivities depend on nucleophilic and general-acid catalysis by cooperative HisH+-His pairs. The peptide catalyst MNV with the HisH+-His pair separated by three residues within the helical segment catalyzes the hydrolysis of p-nitrophenyl fumarate with a second-order rate constant of 0.034 M-1 s-1 at pH 5.1 and 290 K. This i, i+3 site is a factor of three more efficient than the corresponding i, i+4 site. Helix-loop-helix peptides having histidines situated at opposing helices were designed and exhibited cooperative HisH+-His catalytic pairs. The peptide H11,34K hydrolyzed p-nitrophenyl acetate and p-nitrophenyl valerate with second-order rate constants of 0.044 and 0.15 M-1 s-1, respectively, at pH 5.1 and 290 K, indicating that the hydrophobic substituent was recognized by the catalyst.Key words: de novo design, helix-loop-helix, four-helix bundle, histidine, catalysis.