Characterization of the Allosteric Anion-Binding Site of <i>O</i>-Acetylserine Sulfhydrylase

Tai, Chia-Hui; Burkhard, Peter; Gani, David; Jenn, Thierry; Johnson, Corey M.; Cook, Paul F.

doi:10.1021/bi015511s

Cited by 42 publications

(28 citation statements)

References 12 publications

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“…The initial velocity pattern obtained in the absence of added inhibitors exhibits competitive inhibition by both substrates, which is normally diagnostic for a ping-pong mechanism, resulting from substrates binding to the incorrect enzyme form (OAS to F and bisulfide to E). In the present case, however, the inhibition results from the binding of substrates to an inhibitory allosteric site (7).…”

Section: Kinetic Mechanismmentioning

confidence: 72%

Structure and Mechanism of O-Acetylserine Sulfhydrylase

Rabeh

Cook

2004

Journal of Biological Chemistry

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The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a ␤-replacement reaction in which the ␤-acetoxy group of O-acetyl-L-serine (OAS) is replaced by bisulfide to give L-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable ␣-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N-and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for C␤-O bond cleavage than for C␣-H bond cleavage.The biosynthesis of L-cysteine in enteric bacteria, such as Salmonella typhimurium and Escherichia coli, and in plants proceeds via a two-step pathway (Fig. 1). The amino acid precursor of L-cysteine is L-serine, which undergoes a substitution of its ␤-hydroxyl with a thiol in two steps. Serine acetyltransferase (EC 2.3.1.30) catalyzes the acetylation (by acetyl-CoA) of the ␤-hydroxyl of L-serine to give O-acetyl-L-serine (OAS) 1 (1). The final step, the ␣,␤-elimination of acetate from OAS and the addition of H 2 S to give L-cysteine is then catalyzed by Oacetylserine sulfhydrylase (OASS; EC 4.2.99.8). In enteric bacteria, two isozymes of OASS, A and B, are produced under aerobic and anaerobic growth conditions, respectively (2). The A and B isozymes are homodimeric with M r of about 68,900 and 64,000, respectively, and each has one tightly bound pyridoxal 5Ј-phosphate (PLP) per subunit. The structure and mechanism of the A isozyme of OASS from S. typhimurium will be the focus of this minireview. For a more comprehensive review, see Ref.3. It has recently been shown that OASS-A is negatively regulated by small anions binding to an allosteric site at the dimer interface (4), but because of space limitations this aspect will not be discussed in this article. Kinetic MechanismThe kinetic mechanism of OASS is Ping Pong Bi Bi (Fig. 2) as shown by initial velocity studies in the absence and presence of products and dead end inhibitors, isotope exchange at equilibrium, and equilibrium spectral studies (5, 6). O-Acetyl-L-serine binds to the internal aldimine form of the enzyme (E), and acetate is released as the first product. Bisulfide then adds as the second substrate to the ␣-aminoacrylate intermediate form of the enzyme (F), and L-cysteine is released as the final product. The initial velocity pattern obtained in the absence of added inhibitors exhibits competitive inhibition by both substrates, which is normally diagnostic for a ping-pong mechanism, resulting ...

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Section: Kinetic Mechanismmentioning

confidence: 72%

Structure and Mechanism of O-Acetylserine Sulfhydrylase

Rabeh

Cook

2004

Journal of Biological Chemistry

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“…The extinction coefficient at 412 nm of HiOASS, 7600 M −1 cm −1 , was calculated from the amount of PLP released upon alkali‐induced denaturation of the protein fully saturated with PLP (Peterson and Sober 1954). Because chloride ions are known to be allosteric effectors of OASS (Burkhard et al 2000; Tai et al 2001), proteins stock solutions were diluted with or dialyzed against HEPES buffer to reduce the chloride ion concentration.…”

Section: Methodsmentioning

confidence: 99%

Interaction of serine acetyltransferase with O‐acetylserine sulfhydrylase active site: Evidence from fluorescence spectroscopy

et al. 2005

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Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5 0 -phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the a-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.

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“…The pK a value for the H 2 S to SH Ϫ ionization is 7, and experiments were carried out at pH 8 where H 2 S is more than 90% dissociated to bisulfide (43,45). Traces in the presence of bisulfide are invariably noisier than those collected in its absence.…”

Section: Effect Of Cysteine and Bisulfide On The Kinetics Of Formatiomentioning

confidence: 99%

A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

Salsi

Campanini

Bettati

et al. 2010

Journal of Biological Chemistry

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The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

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Characterization of the Allosteric Anion-Binding Site of O-Acetylserine Sulfhydrylase

Cited by 42 publications

References 12 publications

Structure and Mechanism of O-Acetylserine Sulfhydrylase

Structure and Mechanism of O-Acetylserine Sulfhydrylase

Interaction of serine acetyltransferase with O‐acetylserine sulfhydrylase active site: Evidence from fluorescence spectroscopy

A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

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