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Research article - Peer-reviewed, 2009

Protein Autoproteolysis: Conformational Strain Linked to the Rate of Peptide Cleavage by the pH Dependence of the N -> O Acyl Shift Reaction

Johansson, Denny G. A.; Wallin, Goran; Sandberg, Anders; Macao, Bertil; Aqvist, Johan; Hard, Torleif

Abstract

Nucleophilic attack by a side chain nucleophile on the adjacent peptide bond followed by N -> O or N -> S acyl shift is the primary step in protein autoproteotysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteotysis involves substrate destabilization by similar to 7 kcal/mol. However, the precise chemical mechanism by which conformational energy is converted into reaction rate acceleration has not been understood. Here we show that the pH dependence of autoproteolysis in a slow-cleaving mutant (115) of the MUC1 SEA domain is consistent with a mechanism in which N -> O acyl shift proceeds after initial protonation of the amide nitrogen. Unstrained amides have pK(a) values of 0 with protonation on the oxygen, and autoproteotysis is therefore immeasurably slow at neutral pH. However, conformational strain forces the peptide nitrogen into a pyramidal conformation with a significantly increased pK(a) for protonation. We find that pK(a) values of similar to 4 and similar to 6, as in model compounds of twisted amides, reproduce the rate of autoproteolysis in the 1G and wild-type SEA domains, respectively. A mechanism involving strain, nitrogen protonation, and N -> O shift is also supported by quantum-chemical calculations. Such a reaction therefore constitutes an alternative to peptide cleavage that is utilized in autoproteolysis, as opposed to a classical mechanism involving a structurally conserved active site with a catalytic triad and an oxyanion hole, which are not present at the SEA domain cleavage site.

Published in

Journal of the American Chemical Society
2009, Volume: 131, number: 27, pages: 9475–9477

      SLU Authors

    • Härd, Torleif

      • Department of Molecular Biology, Swedish University of Agricultural Sciences
      • University of Gothenburg

    UKÄ Subject classification

    Cell and Molecular Biology

    Publication identifier

    DOI: https://doi.org/10.1021/ja9010817

    Permanent link to this page (URI)

    https://res.slu.se/id/publ/27202