Designed four-helix bundle catalysts--the engineering of reactive sites for hydrolysis and transesterification reactions of p-nitrophenyl esters.
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.
Baltzer
L
L
Department of Chemistry, Göteborg University, Sweden. lars.baltzer@oc.chalmers.se
Broo
K S
KS
Nilsson
H
H
Nilsson
J
J
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004951:Esterification; D004952:Esters / Q000378:metabolism*; D005650:Fumarates / Q000737:chemistry / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010648:Phenylacetates / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
C119041:4-nitrophenyl fumarate; D004952:Esters; D005650:Fumarates; D009578:Nitrobenzenes; D010455:Peptides; D010648:Phenylacetates; D011506:Proteins; D006639:Histidine; D001120:Arginine; D008239:Lysine; C118008:4-nitrophenylacetic acid