Mechanism for intein C-terminal cleavage: a proposal from quantum mechanical calculations

Inteins are autocatalytic protein cleavage and splicing elements. A cysteine to alanine mutation at the N-terminal of inteins inhibits splicing and isolates the C-terminal cleavage reaction. Experiments indicate an enhanced C-terminal cleavage reaction rate upon decreasing the solution pH for the cleavage mutant, which cannot be explained by the existing mechanistic framework. We use intein crystal structure data and the information about conserved amino acids to perform semiempirical PM3 calculations followed by high-level density functional theory calculations in both gas phase and implicit solvent environments. Based on these calculations, we propose a detailed "low pH" mechanism for intein C-terminal cleavage. Water plays an important role in the proposed reaction mechanism, acting as an acid as well as a base. The protonation of the scissile peptide bond nitrogen by a hydronium ion is an important first step in the reaction. That step is followed by the attack of the C-terminal asparagine side chain on its carbonyl carbon, causing succinimide formation and simultaneous peptide bond cleavage. The computed reaction energy barrier in the gas phase is approximately 33 kcal/mol and reduces to approximately 25 kcal/mol in solution, close to the 21 kcal/mol experimentally observed at pH 6.0. This mechanism is consistent with the observed increase in C-terminal cleavage activity at low pH for the cleavage mutant of the Mycobacterium tuberculosis RecA mini-intein.