The influence of protein structure on the products emerging from succinimide hydrolysis.
Proteins are vulnerable to spontaneous, covalent modifications that may result in alterations to structure and function. Asparagines are particularly labile, able to undergo deamidation through the formation of a succinimide intermediate to produce either aspartate or isoaspartate residues. Although aspartates cannot undergo deamidation they can form a succinimide and result in the same products. Isoaspartyls are the principal product of succinimide hydrolysis, accounting for 65-85% of the emerging residues. The variability in the ratio of products emerging from succinimide hydrolysis suggests the ability of protein structure to influence succinimide outcome. In the H15D histidine-containing protein (HPr), phosphorylation of the active site aspartate catalyzes the formation of a cyclic intermediate. Resolution of this species is exclusively to aspartate residues, suggestive of either a succinimide with restrained hydrolysis, or an isoimide, from which aspartyl residues are the only possible product. Deletion of the C-terminal residue of this protein does not influence the ability for phosphorylation or ring formation, but it does allow for isoaspartyl formation, verifying a succinimide as the cyclic intermediate in H15D HPr. Isoaspartyl formation in H15D Delta85 is rationalized to occur as a consequence of elimination of steric restrictions imposed by the C terminus on the main-chain carbonyl of the succinimide, the required point of nucleophilic attack of a water molecule for isoaspartyl formation. This is the first reported demonstration of the influence of protein structure on the products emerging from succinimide hydrolysis.
Athmer
Lance
L
Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
Kindrachuk
Jason
J
Georges
Fawzy
F
Napper
Scott
S
D001224:Aspartic Acid / Q000378:metabolism*; D006868:Hydrolysis; D026581:Isoaspartic Acid / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D013388:Succinimides / Q000378:metabolism*
D026581:Isoaspartic Acid; D011506:Proteins; D013388:Succinimides; C032620:succinimide; D001224:Aspartic Acid