Nucleophilic and general acid catalysis at physiological pH by a designed miniature esterase.
A 31-residue peptide (Art-Est) was designed to catalyse the hydrolysis of p-nitrophenyl esters through histidine catalysis on the solvent exposed face of the alpha-helix of bovine pancreatic polypeptide. NMR spectroscopy indicated that Art-Est adopted a stable 3-dimensional structure in solution. Art-Est was an efficient catalyst with second order rate constants of up to 0.050 M(-1) s(-1). The activity of Art-Est was a consequence of the increased nucleophilicity of His-22, which had a reduced pK(a) value of 5.5 as a consequence of its interaction with His-18 and the positively charged Arg-25 and Arg-26. Mass spectrometry and NMR spectroscopy confirmed that the Art-Est catalysed hydrolysis of p-nitrophenyl esters proceeded through an acyl-enzyme intermediate. A solvent kinetic isotope effect of 1.8 indicated that the transition state preceding the acyl intermediate was stabilised through interaction with the protonated side-chain of His-18 and indicated a reaction mechanism similar to that generally observed for natural esterases. The involvement in the reaction of two histidine residues with different pK(a) values led to a bell-shaped dependence of the reaction rate on the pH of the solution. The catalytic behaviour of Art-Est indicated that designed miniature enzymes can act in a transparent mechanism based fashion with enzyme-like behaviour through the interplay of several amino acid residues.
School of Chemistry, University of Birmingham, Edgbaston, Birmingham, UKB15 2TT.
Andrew J
AJ
Nicoll
Rudolf K
RK
Allemann
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D017433:Protein Structure, Secondary; D013696:Temperature
D004952:Esters; D009596:Nitrophenols; D006639:Histidine; D001120:Arginine; D004950:Esterases