Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternate reactants

Tai, Cheng‐Chi; Nalabolu, Srinivasa; Jacobson, Tony M.; Minter, David E.; Cook, Paul F.

doi:10.1021/bi00076a017

Cited by 115 publications

(189 citation statements)

References 19 publications

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“…Double-reciprocal plots of the initial velocity for the S-sulfo-L-cysteine synthetic reaction were represented as a series of parallel lines. This indicates that the S-sulfo-L-cysteine synthetic reaction followed a typical pingpong bi-bi mechanism, which was identical to the kinetic mechanism of the OAS sulfhydrylation reaction for OASS-As from E. coli and S. enterica serovar Typhimurium (31,41). In this mechanism, OAS first binds to the enzyme to form a Schiff base with PLP, releasing acetic acid.…”

Section: Resultsmentioning

confidence: 64%

Characterization of a Novel Thermostable O -Acetylserine Sulfhydrylase from Aeropyrum pernix K1

Mino

Ishikawa

2003

J Bacteriol

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An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5-phosphate binding motif with both OASS and cystathionine ␤-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100°C. In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.

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Section: Resultsmentioning

confidence: 64%

Characterization of a Novel Thermostable O -Acetylserine Sulfhydrylase from Aeropyrum pernix K1

Mino

Ishikawa

2003

J Bacteriol

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“…Similar mechanisms are utilized by other PLP enzymes that catalyze ␤-elimination and ␤-replacement reactions (28,31), including O-acetylserine sulfhydrylase (32,33), tryptophan indole lyase (34), and tyrosine phenol lyase (35). The reactions in Scheme I include the release and transfer of three different protons.…”

Section: Discussionmentioning

confidence: 92%

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Ro¹,

Miles

1999

Journal of Biological Chemistry

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The mechanism of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK a 1 ‫؍‬ 6.5, pK a 2 ‫؍‬ 7.3, and pK a 3 ‫؍‬ 8.2-9. A significant primary isotope effect (ϳ3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the ␣-proton (␤Lys-87) is protonated at low pH, slowing the abstraction of the ␣-proton and making this step at least partially rate-limiting. pK a 2 is assigned to ␤Lys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK i values of 7.3. The residue with this pK a (␤Lys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK a values support the assignment of pK a 2 to ␤Lys-87 and suggest that the ionizing residue with pK a 1 could be a carboxylate, possibly ␤Asp-305, and that the residue associated with a conformational change at pK a 3 may be ␤Lys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.The tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.1.2.20) is a useful system for investigating relationships between protein structure and function (for reviews, see Refs. 1-4). The separate tryptophan synthase ␤ 2 subunit 1 and the ␤ subunit in the ␣ 2 ␤ 2 complex catalyze the pyridoxal phosphate (PLP) 2 -dependent conversion of L-serine and indole to L-tryptophan. A number of intermediates in this reaction have been identified from spectroscopic and kinetic studies (see Scheme I in Discussion). The three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (5) revealed that the active sites of the ␣ and ␤ subunits are ϳ25 Å apart and are connected by a hydrophobic tunnel. This tunnel serves as a passageway for indole from the active site of the ␣ subunit, where it is produced by cleavage of indole-3-glycerol phosphate, to the active site of the ␤ subunit, where it reacts with L-serine to form L-tryptophan. The activity at each site is modulated by reciprocal communication between the ␣ and ␤ sites. These heterotrophic interactions are proposed to switch the ␣ and ␤ subunits between "open" (catalytically inactive) and "closed" (catalytically active) conformations, to coordinate the activities at the two sites, and to prevent the escape of indole (4). Recent crystallographic results provide direct evidence for ligand-induced conformational changes that result in physical closure of loop structures in the ␣ subunit a...

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“…Second, enteric bacteria and plants synthesize cysteine via direct sulfhydrylation of O-acetylserine by O-acetylserine sulfhydrylase (40), and sulfide is the physiological sulfur donor for this reaction with K m values in the micromolar range (41,42). In contrast, even though sulfide is presumably present at high levels in methanococci, it is probably not the direct sulfur donor for cysteine biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

Cysteine Is Not the Sulfur Source for Iron-Sulfur Cluster and Methionine Biosynthesis in the Methanogenic Archaeon Methanococcus maripaludis

Liu

Sieprawska-Lupa

Whitman

et al. 2010

Journal of Biological Chemistry

111

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Three multiprotein systems are known for iron-sulfur (Fe-S) cluster biogenesis in prokaryotes and eukaryotes as follows: the NIF (nitrogen fixation), the ISC (iron-sulfur cluster), and the SUF (mobilization of sulfur) systems. In all three, cysteine is the physiological sulfur source, and the sulfur is transferred from cysteine desulfurase through a persulfidic intermediate to a scaffold protein. However, the biochemical nature of the sulfur source for Fe-S cluster assembly in archaea is unknown, and many archaea lack homologs of cysteine desulfurases. Methanococcus maripaludis is a methanogenic archaeon that contains a high amount of protein-bound Fe-S clusters (45 nmol/mg protein). Cysteine in this archaeon is synthesized primarily via the tRNA-dependent SepRS/SepCysS pathway. When a ⌬sepS mutant (a cysteine auxotroph) was grown with 34 S-labeled sulfide and unlabeled cysteine, <8% of the cysteine, >92% of the methionine, and >87% of the sulfur in the Fe-S clusters in proteins were labeled, suggesting that the sulfur in methionine and Fe-S clusters was derived predominantly from exogenous sulfide instead of cysteine. Therefore, this investigation challenges the concept that cysteine is always the sulfur source for Fe-S cluster biosynthesis in vivo and suggests that Fe-S clusters are derived from sulfide in those organisms, which live in sulfiderich habitats.

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Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternate reactants

Cited by 115 publications

References 19 publications

Characterization of a Novel Thermostable O -Acetylserine Sulfhydrylase from Aeropyrum pernix K1

Characterization of a Novel Thermostable O -Acetylserine Sulfhydrylase from Aeropyrum pernix K1

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Cysteine Is Not the Sulfur Source for Iron-Sulfur Cluster and Methionine Biosynthesis in the Methanogenic Archaeon Methanococcus maripaludis

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