Sequential site-directed mutagenesis and chemical modification to convert the active site arginine 292 of aspartate aminotransferase to homoarginine

1992 ◽  
Vol 114 (9) ◽  
pp. 3567-3568 ◽  
Author(s):  
Peter W. White ◽  
Jack F. Kirsch
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Aspartate Aminotransferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3–3

1992 ◽  
Vol 286 (1) ◽  
pp. 205-210 ◽  
Author(s):  
W L Chen ◽  
J C Hsieh ◽  
J L Hong ◽  
S P Tsai ◽  
M F Tam
Keyword(s):  
Chemical Modification ◽  
Spodoptera Frugiperda ◽  
Active Site ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Glutathione S Transferase ◽  
Liver Glutathione ◽  
Baculovirus System

Rat liver glutathione S-transferase (GST) 3-3 is composed of two identical subunits, each containing three cysteine residues, Cys-86, Cys-114 and Cys-173. We have shown previously that Cys-86 is not involved in the enzymic activity of GST 3-3 [Hsieh, Huang, Chen, Lai & Tam (1991) Biochem, J. 278, 293-297]. At 50 degrees C, iodoacetamide can inactivate the enzyme by modifying Cys-86 and Cys-114. Cys-114 can be protected against iodoacetamide inhibition by S-(dinitrophenyl)glutathione. Site-directed mutagenesis was used to construct mutants in which serine replaced one (C114S and C173S) or all three (CallS) cysteine residues. These mutants were over-expressed in Spodoptera frugiperda cells in a baculovirus system and were found to be fully active. Replacing Cys-86 or Cys-114 with alanine (C86A and C114A) does not diminish the activity of the protein. The results suggest that cysteines are not involved in the enzymic mechanism, and Cys-114 is possibly located at the active site of GST 3-3.

scholarly journals Identification of an active site cysteine residue in human type I Ins(1,4,5)P3 5-phosphatase by chemical modification and site-directed mutagenesis

1996 ◽  
Vol 320 (1) ◽  
pp. 181-186 ◽  
Author(s):  
David COMMUNI ◽  
Christophe ERNEUX
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type I ◽  
Active Site Cysteine ◽  
Substrate Binding Domain ◽  
Active Site Cysteine Residue

Chemical modification using thiol-directed agents and site-directed mutagenesis have been used to investigate the crucial role of an active site cysteine residue within the substrate-binding domain of human type I Ins(1,4,5)P3 5-phosphatase. Irreversible inhibition of enzymic activity is provoked by chemical modification of the enzyme by N-ethylmaleimide (NEM), 5,5´-dithio-2-nitrobenzoic acid, iodoacetate and to a much smaller extent by iodoacetamide. The alkylation reaction by NEM is prevented in the presence of Ins(1,4,5)P3. The results indicate that NEM binds at the active site of the enzyme with a stoichiometry of 0.9 mol of NEM per mol of enzyme. A single [14C]NEM-modified peptide was isolated after α-chymotrypsin proteolysis of the radiolabelled enzyme and reverse-phase HPLC. Sequence analysis of the active site-labelled peptide (i.e. MNTRCPAWCD) demonstrated that Cys348 contained the radiolabel. Furthermore two mutant enzymes were obtained by site-directed mutagenesis of the cysteine residue to serine and alanine respectively. Both mutant enzymes had identical UV CD spectra. The two mutants (i.e. Cys348 → Ser and Cys348 → Ala) show a marked loss of enzymic activity (more than 98% compared with the wild-type enzyme). Thus we have directly identified a reactive cysteine residue as part of the active site, i.e. the substrate-binding domain, of Ins(1,4,5)P3 5-phosphatase. This cysteine residue is part of a sequence 10 amino acids long that is well conserved among the primary structures of inositol and phosphatidylinositol polyphosphate 5-phosphatases.

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