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Albrecht Messerschmidt - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of Escherichia coli Cystathionine γ‐synthase at 1.5 Å resolution
The EMBO journal, 1998Co-Authors: Tim Clausen, Robert Huber, Markus C. Wahl, Lars Prade, Albrecht MesserschmidtAbstract:The transsulfuration enzyme Cystathionine gamma-synthase (CGS) catalyses the pyridoxal 5'-phosphate (PLP)-dependent gamma-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-Cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of Cystathionine Beta-Lyase (CBL), and refined at 1.5 A resolution to a crystallographic R-factor of 20.0%. The enzyme crystallizes as an alpha4 tetramer with the subunits related by non-crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quaternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate-binding functions of individual active site residues. Enzyme-substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate-binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP-dependent replacement reaction at Cgamma of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.
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Slow-binding inhibition of Escherichia coli Cystathionine Beta-Lyase by L-aminoethoxyvinylglycine: a kinetic and X-ray study.
Biochemistry, 1997Co-Authors: Tim Clausen, Albrecht Messerschmidt, Robert Huber, Hans-dieter Pohlenz, Bernd LaberAbstract:The pyridoxal 5‘-phosphate (PLP)-dependent Cystathionine β-Lyase (CBL) was previously found to be inhibited by the natural toxins rhizobitoxine and l-aminoethoxyvinylglycine (AVG). The present study characterizes the interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) by a combination of kinetic methods and X-ray crystallography. Upon AVG treatment, time-dependent, slow-binding inhibition [Morrison, J. F. (1982) Trends Biochem. Sci. 7, 102−105] was observed due to the generation of a long-lived, slowly dissociating enzyme−inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (CBL + AVG → CBLAVG, Ki = 1.1 ± 0.3 μM) with an association rate constant (k1) of 336 ± 40 M-1 s-1. This value is several orders of magnitude lower than typical bimolecular rate constants of ES formation, suggesting that additional steps occur before formation of the first detectable CBLAVG complex. Loss of activity is paralleled by the conversion of the pyridoxaldimine 426 nm chromophore ...
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Mode of action of Cystathionine Beta-Lyase.
Biological chemistry, 1997Co-Authors: Tim Clausen, Bernd Laber, Albrecht MesserschmidtAbstract:Cystathionine Beta-Lyase (CBL) is a member of the gamma-family of pyridoxal-5'-phosphate (PLP)-dependent enzymes (Alexander et al., 1994) that cleave C(Beta,gamma)-S bonds of a broad variety of substrates. Recently, we reported the X-ray crystal structures of CBL and the CBL-trifluoroalanine inactivation complex at 1.83 A and 2.3 A resolution, respectively. The structures explicitly reveal the cofactor and substrate binding pockets. Spectral analysis of substrate turnover indicates a change of hydrophobicity in the microenvironment of the aldimine bond. In combination with further spectroscopic data, crystallographic evidence permits the formulation of a likely reaction mechanism.
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Crystal structure of the pyridoxal-5'-phosphate dependent Cystathionine Beta-Lyase from Escherichia coli at 1.83 A.
Journal of molecular biology, 1996Co-Authors: Tim Clausen, Bernd Laber, Robert Huber, Hans-dieter Pohlenz, Albrecht MesserschmidtAbstract:Cystathionine Beta-Lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C Beta-S bonds of a broad variety of substrates. The crystal structure of CBL from E. coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data. The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry. A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry. Each monomer of CBL can be described in terms of three spatially and functionally different domains. The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one Beta-strand. It contributes to tetramer formation and is part of the active site of the adjacent subunit. The second domain (residues 61 to 256) harbors PLP and has an alpha/Beta-structure with a seven-stranded Beta-sheet as the central part. The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded Beta-sheet. The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases. Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway. Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes. The structure of the Beta, Beta, Beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2%. It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.
R Douce - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense Cystathionine gamma-synthase cDNA.
Comptes rendus de l’Académie des sciences. Série III Sciences de la vie, 2000Co-Authors: B Gakière, S Ravanel, R Douce, Michel Droux, D JobAbstract:To investigate the role of Cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of Cystathionine Beta-Lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.
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Purification and properties of Cystathionine Beta-Lyase from Arabidopsis thaliana overexpressed in Escherichia coli.
The Biochemical journal, 1996Co-Authors: S Ravanel, D Job, R DouceAbstract:Cystathionine Beta-Lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5'-phosphate-dependent Beta-cleavage of Cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding Cystathionine Beta-Lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis Cystathionine Beta-Lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5'-phosphate. Interaction between the apoenzyme and pyridoxal 5'-phosphate is extremely tight, being characterized by a Kd value of 0.5 microM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant Cystathionine Beta-Lyase resembles that of the enzyme isolated from other sources, Cystathionine and djenkolate being the most effective substrates. The Cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant Cystathionine Beta-Lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5'-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.
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Methionine biosynthesis in higher plants. I. Purification and characterization of Cystathionine gamma-synthase from spinach chloroplasts.
Archives of biochemistry and biophysics, 1995Co-Authors: S Ravanel, M Droux, R DouceAbstract:Cystathionine gamma-synthase, the first enzyme specific for the methionine biosynthetic pathway, was purified to apparent homogeneity from spinach leaf chloroplasts. A nonradioactive assay based on O-phthaldialdehyde derivatization of L-Cystathionine and fluorescence detection was developed to determine the Cystathionine gamma-synthase activity. A unique Cystathionine gamma-synthase activity was located in the stromal fraction of chloroplasts while Cystathionine Beta-Lyase, the second enzyme of the transsulfuration pathway, was associated with both the chloroplastic and cytosolic compartments (see companion manuscript). The purified enzyme exhibited a specific activity of 13 U mg-1. As estimated by gel filtration and polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by activity staining, the native enzyme had an apparent M(r) of 215,000. On the basis of sodium dodecyl sulfate-PAGE, purified Cystathionine gamma-synthase migrated as two molecular species of M(r) 53,000 and 50,000 that are identical in their N-termini. The absorption spectrum obtained at pH 7.5 exhibited a peak at 425 nm due to pyridoxal 5'-phosphate (PLP). The purified enzyme catalyzed the formation of L-Cystathionine or L-homocysteine depending on the sulfur-containing substrate, L-cysteine or sulfide. Maximal Cystathionine gamma-synthase activity was found at pH 7.4. The apparent Km values for O-phospho-L-homoserine (the unique homoserine ester synthesized in the chloroplast), L-cysteine, and sulfide were 1.4, 0.18, and 0.6 mM, respectively. Inactivation of Cystathionine gamma-synthase by DL-propargylglycine (PAG) showed pseudo-first-order kinetics and data were consistent with the existence of an intermediate reversible enzyme-inhibitor complex (Kappi = 140 microM) preceding the formation of a final enzyme-inhibitor complex (kd = 24 x 10(-3) s-1). The irreversibility of the inhibition and the partial restoration of the activity by pyridoxal-phosphate suggest that PAG interacts with the PLP prosthetic group of the enzyme. Kinetic and equilibrium binding studies showed that PAG binding to PLP was considerably enhanced in the enzyme binding pocket compared to that with PLP free in solution.
Tim Clausen - One of the best experts on this subject based on the ideXlab platform.
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Kinetics and inhibition of recombinant human Cystathionine gamma-Lyase. Toward the rational control of transsulfuration.
The Journal of biological chemistry, 1999Co-Authors: Clemens Steegborn, Tim Clausen, Robert Huber, Peter Sondermann, Uwe Jacob, Michael Worbs, Snezan Marinkovic, Markus C. WahlAbstract:The gene encoding human Cystathionine gamma-Lyase was cloned from total cellular Hep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian Cystathionine gamma-Lyase overproduced in a bacterial system. About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with limited success. About 5 mg/liter culture of human Cystathionine gamma-Lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure. While the enzyme showed high gamma-Lyase activity toward L-Cystathionine (Km = 0.5 mM, Vmax = 2.5 units/mg) with an optimum pH of 8.2, no residual Cystathionine Beta-Lyase behavior and only marginal reactivity toward L-cystine and L-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and aminoethoxyvinylglycine. Propargylglycine inactivated human Cystathionine gamma-Lyase much more strongly than trifluoroalanine, in agreement with the enzyme's preference for C-gamma-S bonds. Aminoethoxyvinylglycine showed slow and tight binding characteristics with a Ki of 10.5 microM, comparable with its effect on Cystathionine Beta-Lyase. The results have important implications for the design of specific inhibitors for transsulfuration components.
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Crystal structure of Escherichia coli Cystathionine γ‐synthase at 1.5 Å resolution
The EMBO journal, 1998Co-Authors: Tim Clausen, Robert Huber, Markus C. Wahl, Lars Prade, Albrecht MesserschmidtAbstract:The transsulfuration enzyme Cystathionine gamma-synthase (CGS) catalyses the pyridoxal 5'-phosphate (PLP)-dependent gamma-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-Cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of Cystathionine Beta-Lyase (CBL), and refined at 1.5 A resolution to a crystallographic R-factor of 20.0%. The enzyme crystallizes as an alpha4 tetramer with the subunits related by non-crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quaternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate-binding functions of individual active site residues. Enzyme-substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate-binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP-dependent replacement reaction at Cgamma of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.
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Slow-binding inhibition of Escherichia coli Cystathionine Beta-Lyase by L-aminoethoxyvinylglycine: a kinetic and X-ray study.
Biochemistry, 1997Co-Authors: Tim Clausen, Albrecht Messerschmidt, Robert Huber, Hans-dieter Pohlenz, Bernd LaberAbstract:The pyridoxal 5‘-phosphate (PLP)-dependent Cystathionine β-Lyase (CBL) was previously found to be inhibited by the natural toxins rhizobitoxine and l-aminoethoxyvinylglycine (AVG). The present study characterizes the interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) by a combination of kinetic methods and X-ray crystallography. Upon AVG treatment, time-dependent, slow-binding inhibition [Morrison, J. F. (1982) Trends Biochem. Sci. 7, 102−105] was observed due to the generation of a long-lived, slowly dissociating enzyme−inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (CBL + AVG → CBLAVG, Ki = 1.1 ± 0.3 μM) with an association rate constant (k1) of 336 ± 40 M-1 s-1. This value is several orders of magnitude lower than typical bimolecular rate constants of ES formation, suggesting that additional steps occur before formation of the first detectable CBLAVG complex. Loss of activity is paralleled by the conversion of the pyridoxaldimine 426 nm chromophore ...
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Mode of action of Cystathionine Beta-Lyase.
Biological chemistry, 1997Co-Authors: Tim Clausen, Bernd Laber, Albrecht MesserschmidtAbstract:Cystathionine Beta-Lyase (CBL) is a member of the gamma-family of pyridoxal-5'-phosphate (PLP)-dependent enzymes (Alexander et al., 1994) that cleave C(Beta,gamma)-S bonds of a broad variety of substrates. Recently, we reported the X-ray crystal structures of CBL and the CBL-trifluoroalanine inactivation complex at 1.83 A and 2.3 A resolution, respectively. The structures explicitly reveal the cofactor and substrate binding pockets. Spectral analysis of substrate turnover indicates a change of hydrophobicity in the microenvironment of the aldimine bond. In combination with further spectroscopic data, crystallographic evidence permits the formulation of a likely reaction mechanism.
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Crystal structure of the pyridoxal-5'-phosphate dependent Cystathionine Beta-Lyase from Escherichia coli at 1.83 A.
Journal of molecular biology, 1996Co-Authors: Tim Clausen, Bernd Laber, Robert Huber, Hans-dieter Pohlenz, Albrecht MesserschmidtAbstract:Cystathionine Beta-Lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C Beta-S bonds of a broad variety of substrates. The crystal structure of CBL from E. coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data. The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry. A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry. Each monomer of CBL can be described in terms of three spatially and functionally different domains. The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one Beta-strand. It contributes to tetramer formation and is part of the active site of the adjacent subunit. The second domain (residues 61 to 256) harbors PLP and has an alpha/Beta-structure with a seven-stranded Beta-sheet as the central part. The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded Beta-sheet. The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases. Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway. Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes. The structure of the Beta, Beta, Beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2%. It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.
S Ravanel - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense Cystathionine gamma-synthase cDNA.
Comptes rendus de l’Académie des sciences. Série III Sciences de la vie, 2000Co-Authors: B Gakière, S Ravanel, R Douce, Michel Droux, D JobAbstract:To investigate the role of Cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of Cystathionine Beta-Lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.
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Purification and properties of Cystathionine Beta-Lyase from Arabidopsis thaliana overexpressed in Escherichia coli.
The Biochemical journal, 1996Co-Authors: S Ravanel, D Job, R DouceAbstract:Cystathionine Beta-Lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5'-phosphate-dependent Beta-cleavage of Cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding Cystathionine Beta-Lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis Cystathionine Beta-Lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5'-phosphate. Interaction between the apoenzyme and pyridoxal 5'-phosphate is extremely tight, being characterized by a Kd value of 0.5 microM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant Cystathionine Beta-Lyase resembles that of the enzyme isolated from other sources, Cystathionine and djenkolate being the most effective substrates. The Cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant Cystathionine Beta-Lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5'-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.
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Methionine biosynthesis in higher plants. I. Purification and characterization of Cystathionine gamma-synthase from spinach chloroplasts.
Archives of biochemistry and biophysics, 1995Co-Authors: S Ravanel, M Droux, R DouceAbstract:Cystathionine gamma-synthase, the first enzyme specific for the methionine biosynthetic pathway, was purified to apparent homogeneity from spinach leaf chloroplasts. A nonradioactive assay based on O-phthaldialdehyde derivatization of L-Cystathionine and fluorescence detection was developed to determine the Cystathionine gamma-synthase activity. A unique Cystathionine gamma-synthase activity was located in the stromal fraction of chloroplasts while Cystathionine Beta-Lyase, the second enzyme of the transsulfuration pathway, was associated with both the chloroplastic and cytosolic compartments (see companion manuscript). The purified enzyme exhibited a specific activity of 13 U mg-1. As estimated by gel filtration and polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by activity staining, the native enzyme had an apparent M(r) of 215,000. On the basis of sodium dodecyl sulfate-PAGE, purified Cystathionine gamma-synthase migrated as two molecular species of M(r) 53,000 and 50,000 that are identical in their N-termini. The absorption spectrum obtained at pH 7.5 exhibited a peak at 425 nm due to pyridoxal 5'-phosphate (PLP). The purified enzyme catalyzed the formation of L-Cystathionine or L-homocysteine depending on the sulfur-containing substrate, L-cysteine or sulfide. Maximal Cystathionine gamma-synthase activity was found at pH 7.4. The apparent Km values for O-phospho-L-homoserine (the unique homoserine ester synthesized in the chloroplast), L-cysteine, and sulfide were 1.4, 0.18, and 0.6 mM, respectively. Inactivation of Cystathionine gamma-synthase by DL-propargylglycine (PAG) showed pseudo-first-order kinetics and data were consistent with the existence of an intermediate reversible enzyme-inhibitor complex (Kappi = 140 microM) preceding the formation of a final enzyme-inhibitor complex (kd = 24 x 10(-3) s-1). The irreversibility of the inhibition and the partial restoration of the activity by pyridoxal-phosphate suggest that PAG interacts with the PLP prosthetic group of the enzyme. Kinetic and equilibrium binding studies showed that PAG binding to PLP was considerably enhanced in the enzyme binding pocket compared to that with PLP free in solution.
Philipp Christen - One of the best experts on this subject based on the ideXlab platform.
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Evolutionary relationships among pyridoxal‐5′‐phosphate‐dependent enzymes
European journal of biochemistry, 1994Co-Authors: Frederick W. Alexander, Erika Sandmeier, Perdeep K. Mehta, Philipp ChristenAbstract:Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity. In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named alpha family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-Lyase which, however, catalyze Beta-elimination reactions. The Beta family includes L- and D-serine dehydratase, threonine dehydratase, the Beta subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze Beta-replacement or Beta-elimination reactions. The gamma family incorporates O-succinylhomoserine (thiol-Lyase, O-acetylhomoserine (thiol)-Lyase, and Cystathionine gamma-Lyase, which catalyze gamma-replacement or gamma-elimination reactions, as well as Cystathionine Beta-Lyase. The alpha and gamma family might be distantly related with one another, but are clearly not homologous with the Beta family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the alpha, Beta or gamma family by the criterion of profile analysis:alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B6 enzymes.
