Designed four-helix bundle catalysts--the engineering of reactive sites for hydrolysis and transesterification reactions of p-nitrophenyl esters.

L Baltzer, K S Broo, H Nilsson, J Nilsson

Index: Bioorg. Med. Chem. 7(1) , 83-91, (1999)

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Abstract

Four-helix bundle proteins have been designed that catalyze the hydrolysis and transesterification reactions of p-nitrophenyl esters by a cooperative nucleophilic and general acid mechanism. The catalysts consist of two 42-residue peptides that fold into helix-loop-helix motifs and dimerise. They have previously been shown to recognize anionic and hydrophobic substrates and to follow saturation kinetics. The catalytic entity is a HisH(+)-His pair in a helical segment spaced i, i+4, which can be supplemented by arginines and lysines in the adjacent helix. The binding residues have now been optimized for the catalysis of mono-p-nitrophenyl fumarate hydrolysis and found to vary with the location of the site. The catalytic efficiency of the HisH(+)-His site in helix II in positions 30 and 34 is enhanced by the introduction of arginine and or lysine residues in positions 11 and 15, but not in 8 and 11 or in 15 and 19. The most efficient catalyst using this site, JNIIR11K15, catalyses the reaction with a second-order rate constant of 0.134 M(-1) s(-1) in aqueous solution at pH 5.1 and 290 K. The second-order rate constant is larger than those of the corresponding sites with 'longer' and 'shorter' binding residues. Similar experiments have shown that the efficiency and selectivity of catalysts based on a HisH(+)-11-His-15 site in helix I are enhanced the most by the introduction of Lys-30 and Arg-34.