Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides.

V. Schirch,R. Tyler-Cross

doi:10.1016/s0021-9258(18)54607-x

V. Schirch, R. Tyler-Cross

Open Access

https://doi.org/10.1016/s0021-9258(18)54607-x

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Abstract

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.

Highlights

Amino acids, were determineadt neutral, alkaline, and Clarke, 1987)
We have investigated the rate of 1.0 M NaC1;20 mM CAPS brought to pH 10.0 with NaOH, and 20 deamidation of Asn residues in two series of pentapeptides, VSNXV and VXNSV, where X is one of 10 different amino acids whose side chains vary in charge and size
We describe the char&terization of a peptide bond cleavage reaction which accompanies deamidationin some of the peptides

Summary

There is evidence that in proteins the identityof the nearest

The nonenzymatic deamidationof Asn to Asp is known to occur in proteins and peptides (Clarke, 1985; Wright, 1991a, neighbors of Asn is non-random, suggesting that their selectionis subject toevolutionaryconstraints(Clarke, 1985; Wright, 1991a, 1991b). Model peptide sequences were performed using a series of mM sulfonic acid buffers as follows: usuallyselected onthebasis of amino acidsequences in pH 6.0, MES; pH 7.3, BES; pH 8.0, TAPS; pH 9.0, CHES; and pH proteins which were known t o deamidate (Geiger and Clarke, 1987; Lura and Schirch, 1988; Stephenson and Clarke, 1989; Patel and Borchardt,1990a, 1990b;Robinson etal., 1973).No systematic approach with respect to theeffects of charge and. Reaction products formed during thpeH profile studies of VANSV and the deamidation rates of GANSG, GSNAG, and GSNVG were

EXPERIMENTAL PROCEDURES

RESULTS

VSNRV VSNLV VSNTV VSNIV

VSNTV VSNVV

Buffer or ion

Phosphate Phosphate Phosphate

Deamidation of Asparagine Residues

DISCUSSION