Role of peptide conformation in the rate and mechanism of deamidation of asparaginyl residues.

The tetrapeptides Val-Asn-Gly-Ala and N-acetyl-Val-Asn-Gly-Ala undergo deamidation of the asparaginyl residue at pH 7.0 at similar rates. However, they form different products. The N-acetyl peptide gave a 3:1 ratio of N-acetyl-Val-isoAsp-Gly-Ala and N-acetyl-Val-Asp-Gly-Ala, respectively. The nonacetylated peptide gave no detectable amounts of these products but rather gave a cyclic peptide formed from the nucleophilic displacement of the asparaginyl side chain amide by the amino terminus of valine. This compound was slowly inverted at carbon 2 of the asparaginyl residue. At pH values above 7.5, the nonacetylated peptide also underwent deamidation to form Val-isoAsp-Gly-Ala and Val-Asp-Gly-Ala in the 3:1 ratio. Proton NMR spectra of the acetylated and nonacetylated tetrapeptides show that below pH 7.5 they have very different preferred conformations, and it is these different conformations which result in the different mechanisms of deamidation. Above pH 9.0, both peptides have similar conformations and deamidate by the same mechanism to give equivalent products. Neither mechanism of deamidation was subject to general base catalysis by the buffer. These results suggest that deamidation rates of the asparaginyl-glycyl sequence in proteins will vary according to the conformation of the peptide backbone of each respective protein. The results also show that asparaginyl residues which are penultimate to the amino terminus can react to form an N-terminal-blocked seven-membered ring.

10-Formyltetrahydrofolate synthetase. Evidence for a conformational change in the enzyme upon binding of tetrahydropteroylpolyglutamates.

The 10-formyltetrahydrofolate synthetase domain of the trifunctional enzyme C1-tetrahydrofolate synthase appears to undergo a conformational change in the presence of tetrahydropteroylpolyglutamates, MgATP, and ammonium ion. The binding of these ligands increases the denaturation temperature of the enzyme by 12 degrees C, abolishes the cold lability of the enzyme, and alters its susceptibility to digestion by chymotrypsin. The results suggest that a conformational change is dependent upon binding of the third glutamate residue of tetrahydropteroylpolyglutamates and the beta-phosphoryl group of MgATP. The Km values for MgATP and formate are lowered 3.6- and 520-fold, respectively, when tetrahydropteroyltriglutamate is used as the substrate in place of tetrahydropteroylmonoglutamate. A sensitive coupled assay involving C1-tetrahydrofolate synthase and serine hydroxymethyltransferase was developed to determine the activity of 10-formyltetrahydrofolate synthetase. The assay gives linear rates with the tetrahydropteroylpolyglutamates as substrates but not with the monoglutamate form.