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.

Surface-exposed Tryptophan Residues Are Essential for O-Acetylserine Sulfhydrylase Structure, Function and Stability / Campanini, Barbara; Raboni, Samanta; Vaccari, Simona; Zhang, L.; Cook, P. F.; Hazlett, T. L.; Mozzarelli, Andrea; Bettati, Stefano. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 278:39(2003), pp. 37511-37519. [10.1074/jbc.M305138200]

Surface-exposed Tryptophan Residues Are Essential for O-Acetylserine Sulfhydrylase Structure, Function and Stability

CAMPANINI, Barbara;RABONI, Samanta;VACCARI, Simona;MOZZARELLI, Andrea;BETTATI, Stefano
2003

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.
Surface-exposed Tryptophan Residues Are Essential for O-Acetylserine Sulfhydrylase Structure, Function and Stability / Campanini, Barbara; Raboni, Samanta; Vaccari, Simona; Zhang, L.; Cook, P. F.; Hazlett, T. L.; Mozzarelli, Andrea; Bettati, Stefano. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 278:39(2003), pp. 37511-37519. [10.1074/jbc.M305138200]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/1449877
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