title: Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases.
abstract: Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His(106) and Lys(107) of PmGST B1-1 to investigate their possible role in the enzyme's catalytic activity. His(106) was mutated to Ala, Asn and Phe, and Lys(107) to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His(106) and Lys(107) in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.
authors.affiliation: Dipartimento di Scienze Biomediche, Università 'G. D'Annunzio', Via dei Vestini 31, I-66013 Chieti, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Allocati
authors.forename: E
authors.initials: E
authors.lastname: Casalone
authors.forename: M
authors.initials: M
authors.lastname: Masulli
authors.forename: G
authors.initials: G
authors.lastname: Polekhina
authors.forename: J
authors.initials: J
authors.lastname: Rossjohn
authors.forename: M W
authors.initials: MW
authors.lastname: Parker
authors.forename: C
authors.initials: C
authors.lastname: Di Ilio
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry*; D019791:Guanidine / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011513:Proteus mirabilis / Q000201:enzymology; D012294:Rifamycins / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents; D012294:Rifamycins; D010649:Phenylalanine; D006639:Histidine; D001216:Asparagine; D001120:Arginine; D005982:Glutathione Transferase; D019791:Guanidine; D008239:Lysine; D000409:Alanine