Substitution of Pyridoxal 5′-Phosphate in the O-Acetylserine Sulfhydrylase from Salmonella typhimurium by Cofactor Analogs Provides a Test of the Mechanism Proposed for Formation of the α-Aminoacrylate Intermediate

Cook, Paul F.; Tai, Chia-Hui; Hwang, Chi-Ching; Woehl, Eilika U.; Dunn, Michael F.; Schnackerz, Klaus D.

doi:10.1074/jbc.271.42.25842

Cited by 29 publications

(17 citation statements)

References 27 publications

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“…When PDMP is substituted for PLP in OASS, the reaction with 5-thio-2-nitrobenzoate as substrate showed a V/K TNB value of 31% that of the native enzyme. A slight decrease in the intensity of the internal aldimine in addition to a small red shift is observed for PDMP-OASS in comparison to native OASS (26). Furthermore, the substitution of PLP by PLS yields partially active arginine decarboxylase and tryptophanase (4), but PLS is much less tightly bound than PLP, in agreement with the above assessment.…”

Section: ϫ4supporting

confidence: 76%

“…The PLPMe-DSD, however, shows a significant decrease in the intensity of the visible CD band, suggesting a difference in the orientation of the bound cofactor compared with the other three proteins. The induced CD data for PDMP-OASS and PLPMe-OASS are identical to those of the native enzyme but with a red shift in the max value for the former enzyme (26). In the visible CD, almost identical intensities were observed for native aspartate aminotransferase and PDMP-reconstituted aspartate aminotransferase (52).…”

Section: ϫ4supporting

confidence: 57%

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Substitution of Pyridoxal 5′-Phosphate ind-Serine Dehydratase from Escherichia coliby Cofactor Analogues Provides Information on Cofactor Binding and Catalysis

Schnackerz

Tai

Pötsch

et al. 1999

Journal of Biological Chemistry

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A second low affinity binding site for the cofactor has also been reportedFrom reconstitution experiments of D-serine apodehydratase with various PLP analogues, it was deduced that substitutions at positions 2 and 6 of the coenzyme are not critical for catalytic activity of the dehydratase (3). However, the phosphate group in the 5Ј-position of the cofactor in its dianionic form is essential (4, 5). The absorption spectrum of highly purified DSD exhibits a prominent absorbance maximum at 415 nm, indicating a protonated Schiff base linkage of the formyl group of PLP to the ⑀-amino group of Lys-118 (1, 6, 7). Excitation of D-serine dehydratase at 296 nm causes an emission spectrum with maxima at 335 and 510 nm, respectively (8 -11). The emission maximum at 510 nm is due to energy transfer between a tryptophanyl residue and PLP (9). The dehydratase is weakly fluorescent with an excitation maximum at 415 nm corresponding to the absorbance maximum of the internal Schiff base and an emission maximum around 510 nm (8 -11), typical of all PLP-dependent enzymes studied so far (12-16). Upon addition of 0.5 M glycine or L-alanine the fluorescence intensity at 510 nm increased markedly, and the max shifts from 510 to 485 (9, 10), similar to spectral changes observed for O-acetylserine sulfhydrylase (12). As suggested above a particularly useful approach to investigate the active sites of PLP enzymes is measuring spectral properties associated with the protein and cofactor. In this study we have investigated the environment of the natural cofactor and three cofactor analogues, one active and two inactive, in DSD by CD and fluorescence spectroscopy. EXPERIMENTAL PROCEDURESChemicals-PDMP was kindly provided by Dr. O. Saiko (Merck). PLP monomethyl ester, prepared by the method of Pfeuffer et al. (17), was a gift of Dr. J. Ehrlich. Pyridoxal 5Ј-sulfate was prepared by the method of Kuroda (18). All other chemicals were of highest quality commercially available.Enzymes-D-Serine dehydratase was purified from Escherichia coli K12 mutant C6 as described by Schiltz and Schnackerz (19) or from a wild-type DSD expression plasmid (11). D-Serine apodehydratase was prepared by using the resolution procedure described by Dowhan and Snell (1). The apoenzyme had a residual activity of 1.9 units/mg of protein. The specific activity of PLP-reconstituted dehydratase was 100 units/mg of protein when measured at 25°C. Reconstitution of apodehydratase with PLP analogues was achieved by incubating apoenzyme with a 5-fold excess of the respective cofactor analogue for 1 h at 25°C in the dark. Excess cofactor analogue was removed by passing the incubation mixture over a Sephadex G-25 (3.4 ϫ 40 cm) or PD10 column equilibrated with 100 mM potassium phosphate buffer, pH 7.8. The specific activity of the PDMP-reconstituted dehydratase was 38 units/mg of protein.Enzyme Assay-The enzymatic activity of DSD was determined at 25°C as described by Dowhan and Snell (1). The assay mixture contains * This work was supported in part by funds from the Deutsche For...

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Section: ϫ4supporting

confidence: 76%

Section: ϫ4supporting

confidence: 57%

Substitution of Pyridoxal 5′-Phosphate ind-Serine Dehydratase from Escherichia coliby Cofactor Analogues Provides Information on Cofactor Binding and Catalysis

Schnackerz

Tai

Pötsch

et al. 1999

Journal of Biological Chemistry

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“…Ultraviolet-visible spectral studies are consistent with an internal Schiff base ( max ϭ 412 nm) present as the resting form of the enzyme and the ␣-aminoacrylate external Schiff base ( max ϭ 330, 470 nm) present after elimination of the ␤-acetoxy group (5,10,11,14).…”

Section: Chemical Mechanismmentioning

confidence: 71%

“…In a classical one-site ping-pong mechanism, the individual half-reactions are independent of the concentration of the other substrate. This is not the case for OASS, and the V/K OAS values were not identical when bisulfide (10 5 M Ϫ1 s Ϫ1 ) or 5-thio-2-nitrobenzoate (TNB; 37 M Ϫ1 s Ϫ1 ) was the nucleophilic substrate (11). The V/K values for the second half-reaction were within a factor of 2 of one another when different amino acid substrates were used.…”

mentioning

confidence: 77%

“…4) (9). The 5Ј-phosphate of the cofactor is dianionic as suggested by 31 P NMR (10,11), and its charge is partially neutralized by the dipole of helix 7 in the C-terminal domain, one of three helix dipoles directed toward the active site. The phenolic O-3Ј of the cofactor is within hydrogen-bonding distance of the amide nitrogen of the Asn-71 side chain and the Schiff base nitrogen.…”

Section: Structure Of Oass-amentioning

confidence: 98%

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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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