A structural and functional Analysis

Pyridoxial 5'-phosphate (PLP) acts as a cofactor in many enzymes involved in diverse aspects of amino acid metabolism such as transamination, β/γ-elimination, β/γ-replacement and racemization. In all PLP-dependant enzymes the carbonyl group of the PLP coenzyme binds to an ε-amino group of a lysine residue in the active site, forming an internal aldimine. O-Acetylserine sulfahydrylase (OASS), isolated from Salmonella typhimurium, belongs to the β -family of PLP-dependant enzymes and catalyzes the last step in the cysteine biosynthesis pathway via β-replacement, converting O-acetylserine (OAS) to cysteine, upon exchanging acetate in the OAS side chain for sulfide (Fig. 1). The structural and functional framework underlying the reaction mechanism for OASS has been characterized extensively by kinetic studies, site-directed mutagenesis, UV-visible fluorescence and phosphorescence spectroscopy and x-ray structural determination. Three conformationally distinct "open", "closed", and "inhibited" states were elucidated.

Understanding the organization of secondary, tertiary and quaternary structural elements is crucial to characterizing a protein's function. While primary amino acid sequence can be used to predict secondary motifs, the sequence alone yields limited information. Tryptophan synthase β (TRPS β), for example, has only 19% amino acid sequence similarity to OASS, yet all the secondary structural elements in OASS are found in TRPS β in equivalent order and spatial arrangement (Fig. 2). The structural equivalence reflects similar function, for both are PLP-dependent enzymes that catalyze β-replacement reactions in amino acid biosynthesis; TRPS β converts L-serine to L-tryptophan.

The large shift in Asn69 seems to induce larger conformational changes in a subdomain including 87-113. This subdomain is part of the N-terminus domain and comprises β-strand 4, α-helix 3, β-strand 5 and α-helix 4. The relationship between the local and global rearrangements induced upon substrate binding must be further investigated, however, the following series of events have been inferred by analyzing the positions of all residues in both the open and closed conformations. The dramatic movement of side chain Asn69 leaves a hole within the protein. The side chain of Met95, which is situated adjacent to Asn69, moves along with it, filling the space. The sulfur atom of Met95 side chain moves 6.5 Å. Met95 is part of the movable subdomain, 87-113, which then shifts as a rigid body (Fig. 9). The shift brings residues of the N domain close enough to the C-terminal domain for hydrophobic interactions to further stabilize the new position (Table 1). Burkhard et al., have envisioned possible functions for the observed changes assuming OAS occupies the same orientation as the Met substrate analog. In the closed conformation functional groups become properly positioned to facilitate the β-elimination reaction of acetic acid from the OAS side chain, which is thought to undergo a concerted E2-type reaction (Hwang et al., 1996). Furthermore elimination yields the α-aminoacrylate intermediate, which is highly reactive and should be unstable. The restriction of the bulk solvent to the active site would prevent the destabilization of the reactive intermediate. As seen in figure 9, the closed conformation has only a narrow channel to the active site, permitting passage only to small molecules such as the acetate leaving group and the SH- second substrate. Evidence that the external aldimine linkage between PLP and Met can be reduced by sodium [H3] borohydride in the native conformation and not in the mutant K41A supports the hypothesis that the reactive intermediate is protected from bulk solvent in the closed state (Strambini et al., 1996). This type of conformational change resulting from substrate binding is called induced fit. This dynamic mechanism is allows efficient substrate binding as well as protection from the bulk solvent and is often essential to enzymatic function. The proteins ability to make the conformational change is built in to the structure of the protein at all four levels of organization so that the conversion is energetically favorable.

Inhibited Conformation Resulting from Local and Global Conformational Changes.

OASS is a homodimeric protein, whereby each single chain monomer is composed of two domains, the N-terminus and the C-terminus. Both domains have a central β-sheet surrounded by several alpha helixes (Fig. 3 and Fig 4). The N and C domains together participate in the homodimeric interaction between the two identical fully folded OASS chains, consisting of 1354 Å2 buried per monomer. Both polar and nonpolar interactions result in enthalpic and entropic conditions favoring dimerization. One interface ion pair between oppositely charged side chains (Glu 303 of one monomer and Arg 304 of the other monomer) contribute to dimmer stability. This int

Some common words found in the essay are:
Proximity Pro36, Protein Folding, Conformational Changes, N-terminus C-terminus, Gly178 Thr180, McClure Cook, Dynamic AOSS, Concluding Remarks, Topology Understanding, SH- Catalytic, et al, burkhard et al, active site, burkhard et, et al 1996, al 1996, closed conformation, schiff base, conformational changes, free energy, amino acid, inhibited conformation, schiff base linkage, et al 2000, et al 1993,
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