title: Protein autoproteolysis: conformational strain linked to the rate of peptide cleavage by the pH dependence of the N --> O acyl shift reaction.
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.
authors.affiliation: Department of Medical Biochemistry and Swedish NMR Centre, University of Gothenburg, Box 440, SE-405 30 Göteborg, Sweden.
authors.forename: Denny G A
authors.initials: DG
authors.lastname: Johansson
authors.forename: Göran
authors.initials: G
authors.lastname: Wallin
authors.forename: Anders
authors.initials: A
authors.lastname: Sandberg
authors.forename: Bertil
authors.initials: B
authors.lastname: Macao
authors.forename: Johan
authors.initials: J
authors.lastname: Aqvist
authors.forename: Torleif
authors.initials: T
authors.lastname: Härd
mesh_terms: D006863:Hydrogen-Ion Concentration; D009584:Nitrogen / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011789:Quantum Theory
chemical_list: D010455:Peptides; D011506:Proteins; D009584:Nitrogen; D010100:Oxygen