Versatility of helix-loop-helix catalysts - cooperative HisH(+)-His sites that span both helices

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 nucleophi lic and general-acid catalysis by cooperative HisH(+)-His pairs. The peptid e catalyst MNV with the HisH(+)-His pair separated by three residues within the helical segment catalyzes the hydrolysis of p-nitrophenyl fumarate wit h 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 va lerate with second-order rate constants of 0.044 and 0.15 M-1 s(-1), respec tively, at pH 5.1 and 290 K, indicating that the hydrophobic substituent wa s recognized by the catalyst.