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Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase.
Biochemistry. 2005 Mar 08; 44(9):3636-43.B

Abstract

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.

Authors+Show Affiliations

Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15736973

Citation

Paradisi, Francesca, et al. "Spontaneous Chemical Reversion of an Active Site Mutation: Deamidation of an Asparagine Residue Replacing the Catalytic Aspartic Acid of Glutamate Dehydrogenase." Biochemistry, vol. 44, no. 9, 2005, pp. 3636-43.
Paradisi F, Dean JL, Geoghegan KF, et al. Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase. Biochemistry. 2005;44(9):3636-43.
Paradisi, F., Dean, J. L., Geoghegan, K. F., & Engel, P. C. (2005). Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase. Biochemistry, 44(9), 3636-43.
Paradisi F, et al. Spontaneous Chemical Reversion of an Active Site Mutation: Deamidation of an Asparagine Residue Replacing the Catalytic Aspartic Acid of Glutamate Dehydrogenase. Biochemistry. 2005 Mar 8;44(9):3636-43. PubMed PMID: 15736973.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase. AU - Paradisi,Francesca, AU - Dean,Jonathan L E, AU - Geoghegan,Kieran F, AU - Engel,Paul C, PY - 2005/3/2/pubmed PY - 2005/5/7/medline PY - 2005/3/2/entrez SP - 3636 EP - 43 JF - Biochemistry JO - Biochemistry VL - 44 IS - 9 N2 - A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15736973/Spontaneous_chemical_reversion_of_an_active_site_mutation:_deamidation_of_an_asparagine_residue_replacing_the_catalytic_aspartic_acid_of_glutamate_dehydrogenase_ DB - PRIME DP - Unbound Medicine ER -