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Protein autoproteolysis: conformational strain linked to the rate of peptide cleavage by the pH dependence of the N --> O acyl shift reaction.

Journal article
Authors Denny Johansson
Göran Wallin
Anders Sandberg
Bertil Macao
Johan Aqvist
Torleif Härd
Published in Journal of the American Chemical Society
Volume 131
Issue 27
Pages 9475-7
ISSN 1520-5126
Publication year 2009
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 9475-7
Language en
Links dx.doi.org/10.1021/ja9010817
Keywords Hydrogen-Ion Concentration, Nitrogen, chemistry, Oxygen, chemistry, Peptides, chemistry, Protein Conformation, Proteins, chemistry, Quantum Theory
Subject categories Biophysical chemistry, Biochemistry

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 autoproteolysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteolysis involves substrate destabilization by approximately 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 (1G) 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 autoproteolysis 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 approximately 4 and approximately 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.

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