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Issue 15, 2004
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Nucleophilic and general acid catalysis at physiological pH by a designed miniature esterase

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Abstract

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 α-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 pKa 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 pKa 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.

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

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Publication details

The article was received on 30 Mar 2004, accepted on 14 Jun 2004 and first published on 08 Jul 2004


Article type: Paper
DOI: 10.1039/B404730C
Org. Biomol. Chem., 2004,2, 2175-2180

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

    A. J. Nicoll and R. K. Allemann, Org. Biomol. Chem., 2004, 2, 2175
    DOI: 10.1039/B404730C

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