Spontaneous Chemical Reversion of an Active Site Mutation: Deamidation of an Asparagine Residue Replacing the Catalytic Aspartic Acid of Glutamate Dehydrogenase
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- 作者：Francesca Paradisi ; Jonathan L. E. Dean ; Kieran F. Geoghegan ; and Paul C. Engel
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：March 8, 2005
- 年：2005
- 卷：44
- 期：9
- 页码：3636 - 3643
- 全文大小：134K
- 年卷期：v.44,no.9(March 8, 2005)
- ISSN：1520-4995
文摘
A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Aspis replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expressionin Escherichia coli at 37 
f">C. This low-level activity also displayed Michaelis constants for substrates thatwere remarkably similar to those of the wild-type enzyme. Expression at 8 f">C gave a mutant enzymepreparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubationat 37 f">C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggestedthat the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptidesderived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDHfreshly prepared at 8 f">C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-weekincubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainlyoccurred in sample workup rather than during the 2-week incubation; (iv) that there is no significantdeamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) thatwhen the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears thatthis deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literaturesearch indicates that this finding is not unique and that Asn may not be a suitable mutational replacementin the assessment of putative catalytic Asp residues by site-directed mutagenesis.
f">C. This low-level activity also displayed Michaelis constants for substrates thatwere remarkably similar to those of the wild-type enzyme. Expression at 8 f">C gave a mutant enzymepreparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubationat 37 f">C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggestedthat the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptidesderived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDHfreshly prepared at 8 f">C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-weekincubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainlyoccurred in sample workup rather than during the 2-week incubation; (iv) that there is no significantdeamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) thatwhen the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears thatthis deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literaturesearch indicates that this finding is not unique and that Asn may not be a suitable mutational replacementin the assessment of putative catalytic Asp residues by site-directed mutagenesis.