Abstract
Proteins utilizing pyridoxal 5′-phosphate as a coenzyme constitute a large superfamily and are currently classified into three functional groups and five structural fold types. Despite the variability of sequences and catalyzed reactions, they share relevant structural, dynamic and functional properties. Therefore, they constitute an optimal system to investigate the relative influence of primary sequence and coenzyme interactions on folding pathways, structural stability and enzymatic function. O-Acetylserine sulfhydrylase is a dimeric pyridoxal 5′-phosphate dependent enzyme that catalyzes the synthesis of l-cysteine from O-acetylserine and sulfide. The time-resolved fluorescence study of O-acetylserine sulfhydrylase unfolding, here reported, indicates that the coenzyme stabilizes the protein structure. The dependence on denaturant concentration of tryptophan lifetimes in the holo- and apo-enzyme demonstrates that the interactions with the coenzyme stabilize the C-terminal domain to a higher extent with respect to the N-terminal domain. This result is discussed in terms of a linkage between the differential stabilization brought about by the coenzyme and the different degrees of conformational flexibility required by the specialized functional role of distinct protein regions.
Highlights
O-Acetylserine sulfhydrylase (OASS) is a homodimeric enzyme (34 450 Da/protomer for the Salmo-Abbreviations: PLP, pyridoxal 5P-phosphate; OASS, O-acetylserine sulfhydrylase; TS, tryptophan synthase; NATA, N-acetyltryptophan-amide; Trp, tryptophan; GdnHCl, guanidinium hydrochloride; HEPES, N-2-hydroxyethylpiperazine-NP-2ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid nella typhimurium enzyme) catalyzing the last step of cysteine biosynthesis in bacteria and plants
The total £uorescence intensity upon coenzyme £uorescence excitation at 454.5 nm drops sharply with the increase of GdnHCl concentration, and reliable measurements could not be carried out above 2.0 M. These results are in agreement with absorption and 31P nuclear magnetic resonance data [12] and indicate that most of the coenzyme is released from the protein upon exposure to 2.0 M GdnHCl
On the basis of previous studies on native holo-OASS [10], the long (Fig. 2, circles) and short-lived components (Fig. 2, squares) have been attributed to the ketoenamine tautomer of the internal aldimine and to a dipolar species occurring upon imine proton dissociation in the excited state, respectively
Summary
O-Acetylserine sulfhydrylase (OASS) is a homodimeric enzyme The structural and functional similarities within and across the protein subgroups often exist with low sequence homologies, as in the case of tryptophan synthase (TS) and OASS, both belonging to the L-family and fold type II This feature makes PLP-dependent enzymes ideal systems in which to investigate the delicate interplay between the primary sequence and the coenzyme-mediated e¡ects in determining the folding mechanism, functional structure and global and local stability of the native conformation(s). Since specialized regions within each macromolecule ful¢ll speci¢c tasks in protein function and regulation, it is reasonable to assume that individual domains are endowed with di¡erent stability depending on the extent of conformational £exibility required for biological functions It is of considerable interest (i) to assess the relative thermodynamic stability of the N- and C-terminal domains of OASS and (ii) to relate it to the dynamics associated to protein function and regulation
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