Kinetic Isotope Effects as a Probe of the β-Elimination Reaction Catalyzed by <i>O</i>-Acetylserine Sulfhydrylase

Hwang, Chi-Ching; Woehl, Eilika U.; Minter, David E.; Dunn, Michael F.; Cook, Paul F.

doi:10.1021/bi9602472

Cited by 34 publications

(39 citation statements)

References 21 publications

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“…This is consistent with the lack of a primary kinetic isotope effect on aminoacrylate formation in yCBS (25). In contrast, a primary kinetic isotope effect on formation of an aminoacrylate intermediate is seen in both O-acetylserine sulfhydrylase and tryptophan synthase, which are fold II PLP enzymes that catalyze ␤-replacement reactions (20,29,30).…”

Section: Discussionsupporting

confidence: 76%

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Yadav

Banerjee

2012

Journal of Biological Chemistry

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Section: Discussionsupporting

confidence: 76%

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Yadav

Banerjee

2012

Journal of Biological Chemistry

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“…The primary deuterium isotope effect measured using OAS-2D in the steady state is pH-dependent, increasing from a value of 1.7 at neutral pH to a limiting value of 2.8 at low pH (8). Data indicate that OAS is a sticky substrate with a stickiness factor of 1.5, that is once OAS binds to enzyme it goes on to produce the ␣-aminoacrylate intermediate 1.5 times faster than it dissociates from enzyme.…”

Section: Minireview: Mechanism Of O-acetylserine Sulfhydrylase 26805mentioning

confidence: 96%

“…The second half-reaction is very fast and likely diffusion limited with a first-order rate constant of Ͼ1000 s Ϫ1 measured at 5 M bisulfide at pH 6.5 (8). In a classical one-site ping-pong mechanism, the individual half-reactions are independent of the concentration of the other substrate.…”

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confidence: 99%

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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“…The N-terminal domain (residues 46 -153) contains a parallel ␤-sheet flanked by three ␣-helices on one side, two of which are part of the dimer interface, and one ␣-helix located on the opposite side of the sheet forming part of the active site entrance. The C-terminal domain consists of residues 11-45, including a unique insertion of eight amino acids (27)(28)(29)(30)(31)(32)(33)(34), and residues 154 -323. This domain is built up by a six-stranded mixed ␤-sheet sandwiched by four ␣-helices, two on each side.…”

Section: Characterization Of M Tuberculosis Cysm-recombinantmentioning

confidence: 99%

Cysteine Synthase (CysM) of Mycobacterium tuberculosis Is an O-Phosphoserine Sulfhydrylase

Ågren

Schnell

Oehlmann

et al. 2008

Journal of Biological Chemistry

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The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-Å resolution.

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Kinetic Isotope Effects as a Probe of the β-Elimination Reaction Catalyzed by O-Acetylserine Sulfhydrylase

Cited by 34 publications

References 21 publications

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Structure and Mechanism of O-Acetylserine Sulfhydrylase

Cysteine Synthase (CysM) of Mycobacterium tuberculosis Is an O-Phosphoserine Sulfhydrylase

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