Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure, function, and stability.
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.
Campanini
Barbara
B
Department of Biochemistry and Molecular Biology, University of Parma, Italy.
Raboni
Samanta
S
Vaccari
Simona
S
Zhang
Lei
L
Cook
Paul F
PF
Hazlett
Theodore L
TL
Mozzarelli
Andrea
A
Bettati
Stefano
S
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
D020106:Acrylamide; D014364:Tryptophan; D003547:Cysteine Synthase