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.
Highlights
O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism
Absorbance Spectra of W50Y and W161Y Mutants—The UVvisible spectra of the single tryptophan mutants are qualitatively similar to the spectrum of the WT protein, showing two peaks centered at 278 and 412 nm (Fig. 2)
The W50Y mutant shows a band at 500 nm, which exhibits an intensity about half of that of the wild type protein, indicating the occurrence of an energy transfer process between Trp161 and pyridoxal 5Ј-phosphate (PLP)
Summary
O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. 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 biosynthesis of cysteine in bacteria and plants is accomplished by the pyridoxal 5Ј-phosphate (PLP)1-dependent enzyme O-acetylserine sulfhydrylase (OASS). PLP-dependent enzymes are currently classified into three functional families, depending on the mechanism of the catalyzed reaction, and into 5-fold types, depending on the structural arrangement. OASS possesses two tryptophan residues: Trp in the Nterminal domain and Trp161 in the C-terminal domain. The aim of the present work was to characterize the role of the tryptophans in the structure, function, and stability of OASS and to gain insight into the unfolding processes of the Nand C-terminal domains
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