Selective deamidation of ribonuclease A. Isolation and characterization of the resulting isoaspartyl and aspartyl derivatives.
Selective deamidation of proteins and peptides is a reaction of great interest, whether it has physiological significance as in protein aging, or occurs as a disturbing event in the preparation of natural or recombinant proteins. Deamidation of bovine pancreatic ribonuclease A, RNase A, a classical model protein, has been reported to occur only after denaturation of the protein, or under harsh conditions. In this paper convenient procedures are described for selective deamidation of Asn67 in native RNase A under mild conditions. Furthermore, for the first time, both products of deamidation were isolated: the aspartyl and the isoaspartyl containing protein derivatives. Replacement of Asn67 with either residue lowers the catalytic activity of the enzyme, on RNA and on model substrates, except when a dinucleotide with a purine on the 5' side is the substrate. In the latter case an intriguing increase in the specificity constant is observed. The Asp67 derivative was found to refold, after full denaturation and reduction, at the same rate as the fully amidated protein, whereas the iso-Asp67 derivative refolded at half that rate. It is hypothesized that this effect is due to a delayed formation of disulfide 65-72 for the presence of the abnormal isopeptide bond between residues 67 and 68.
Di Donato
A
A
Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, Naples, Italy.
Ciardiello
M A
MA
de Nigris
M
M
Piccoli
R
R
Mazzarella
L
L
D'Alessio
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003641:Deamination; D007700:Kinetics; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010449:Peptide Mapping; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification
D001224:Aspartic Acid; D012259:Ribonuclease, Pancreatic