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

Nicoll, Andrew J. and Allemann, Rudolf Konrad 2004. Nucleophilic and general acid catalysis at physiological pH by a designed miniature esterase. Organic & Biomolecular Chemistry 2 (15) , pp. 2175-2180. 10.1039/b404730c

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Cardiff Catalysis Institute (CCI)
Subjects: Q Science > QD Chemistry
Publisher: RSC Publishing
ISSN: 1477-0520
Last Modified: 04 Jun 2017 02:54
URI: http://orca.cf.ac.uk/id/eprint/13487

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