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J. Biol. Chem., Vol. 278, Issue 39, 37511-37519, September 26, 2003

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

Barbara Campanini  , Samanta Raboni  , Simona Vaccari , Lei Zhang ¶, Paul F. Cook ¶, Theodore L. Hazlett || **, Andrea Mozzarelli   and Stefano Bettati   

From the Department of Biochemistry and Molecular Biology, the National Institute for the Physics of Matter, and the Department of Public Health, University of Parma, Parma 43100, Italy, the ^¶Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, and the ^||Laboratory of Fluorescence Dynamics, University of Illinois, Urbana, Illinois 61801

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.

Received for publication, May 16, 2003 , and in revised form, June 13, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Supported by National Institutes of Health Grant RR03155.

To whom correspondence should be addressed. Tel.: 39-0521-903721; Fax: 39-0521-903712; E-mail: stefano.bettati{at}unipr.it.

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