title: Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure, function, and stability.
abstract: O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of the accessibility of the active site to acrylamide and stabilizes thermodynamically the N-terminal domain, a result consistent with stronger interdomain interactions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Parma, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.forename: Samanta
authors.initials: S
authors.lastname: Raboni
authors.forename: Simona
authors.initials: S
authors.lastname: Vaccari
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
authors.forename: Theodore L
authors.initials: TL
authors.lastname: Hazlett
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
authors.forename: Stefano
authors.initials: S
authors.lastname: Bettati
mesh_terms: D020106:Acrylamide / Q000494:pharmacology; D001665:Binding Sites; D002942:Circular Dichroism; D003547:Cysteine Synthase / Q000737:chemistry* / Q000502:physiology; D004795:Enzyme Stability; D005453:Fluorescence; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014364:Tryptophan
chemical_list: D020106:Acrylamide; D014364:Tryptophan; D003547:Cysteine Synthase