Reactive-site design in folded-polypeptide catalysts - The leaving group pK(a) of reactive esters sets the stage for cooperativity in nucleophilic and general-acid catalysis
2000 (English)In: Chemistry - A European Journal, ISSN 0947-6539, Vol. 6, no 12, 2214-2220 p.Article in journal (Refereed) Published
The second-order rate constants for the hydrolysis of nitrophenyl esters catalysed by a number of folded designed polypeptides have been determined. and 1900-fold rate enhancements over those of the 4-methylimidazole-catalysed reactions have been observed. The rate enhancements are much larger than those expected from the pK(a) depression of the nucleophilic His residues alone. Kinetic solvent isotope effects were observed at pn values lower than the pK(a), values of the leaving groups and suggests that general-acid catalysis contributes in the pH range where the leaving group is predominantly protonated. In contrast, no isotope effects were observed at pH values above the pK(a) of the leaving group. A Hammett rho value of 1.4 has been determined fur the peptide-catalysed hydrolysis reaction by variation of the substituents of the leaving phenol. The corresponding values For the imidazole-catalysed reaction is 0.8 and For phenol dissociation is 2.2. There is therefore, very approximately, half a negative charge localised on the phenolate oxygen in the transition stair in agreement with the conclusion that transition-state hydrogen-bond formation may contribute to the observed catalysis. The elucidation at a molecular level of the principles that control cooperativity in the biocatalysed ester-hydrolysis reaction represents the first step towards a level of understanding of the concept of cooperativity that may eventually allow us to design tailor-made enzymes for chemical reactions not catalysed by nature.
Place, publisher, year, edition, pages
2000. Vol. 6, no 12, 2214-2220 p.
catalysts, de novo design, helical structures, histidine
Engineering and Technology
IdentifiersURN: urn:nbn:se:liu:diva-49695OAI: oai:DiVA.org:liu-49695DiVA: diva2:270591