Nucleophilic and general acid catalysis at physiological pH by a designed miniature esterase

Org Biomol Chem. 2004 Aug 7;2(15):2175-80. doi: 10.1039/B404730C. Epub 2004 Jul 8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arginine / chemistry
  • Arginine / metabolism
  • Catalysis
  • Cattle
  • Esterases / chemistry
  • Esterases / genetics
  • Esterases / metabolism*
  • Esters / chemistry
  • Esters / metabolism
  • Histidine / chemistry
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Nitrophenols / chemistry
  • Nitrophenols / metabolism
  • Pancreas / metabolism
  • Protein Structure, Secondary
  • Temperature


  • Esters
  • Nitrophenols
  • Histidine
  • Arginine
  • Esterases