Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.     

Mechanistic studies with vinylglycine and beta-haloaminobutyrates as substrates for cystathionine gamma-synthetase from Salmonella typhimurium.

Cystathionine gamma-synthetase (EC 4.2.99.9), a key enzyme in bacterial methionoine biosynthesis, has been found to use L-vinylglycine (2-amino-3-butenoate) and L-beta-haloaminobutyrates (X = F, Cl) as substrates in addition to the physiological gamma-substituted substrate O-succinyl-L-homoserine (OSHS). Vinylglycine is a substrate both for alpha-ketobutyrate formation (the normal product from gamma elimination with OSHS) and for cystathionine formation (the normal gamma-replacement product with OSHS) in the presence of cysteine. This behavior substantiates that the stabilized vinylglycine--pyridoxal phosphate (PLP) alpha carbanion is the key partitioning species in this enzyme's catalysis. The Vmax values for ketobutyrate production and cystathonine formation from vinylglycine are equivalent at approximately 45 U/mg, whereas the corresponding Vmax values from OSHS are 20 and 200 U/mg, respectively, suggesting different rate-determining steps with these two substrates. The beta-haloaminobutyrates undergo catalyzed HX elimination to yield bound aminocrotonate--PLP directly as a an initial intermediate and as a precursor of ketobutyrate. Little or no cystathionine formation is detectable when these substrates are incubated with enzyme and the normal cosubstrate cysteine, strongly indicating that the aminocrotonate--PLP intermediate is not in rapid, reversible equilibrium with the stabilized vinylglycine--PLP carbanion; in normal catalysis, the prototropic shift from alpha carbanion to aminocrotonate appears functionally unidirectional. The HX-elimination step from beta-chloroaminobutyrate is nonconcerted as demonstrated by a 3H2O in equilibrium chloroaminobutyrate exchange reaction. Further suggestion for discrete beta-halo-alpha-carbanionic intermediates derives from the observation that the haloaminobutyrates appear to a partition between ketobutyrate formation and enzyme inactivation. Since neither vinylglycine nor OSHS causes any detectable inactivation during turnover, it is likely that the inactivation species is not a common intermediate, i.e., the electrophilic aminocrotonate--PLP species (a potential Michael acceptor), but rather a species peculiar to the beta-haloaminobutyrate pathway. The beta-halo-alpha-carbanion--PLP intermediate has beta-halo-alpha-iminodihydropyridine character in the p-quinoid resonance contributor and is a good candidate for an alkylating agent by an SN2--displacement mechanism. Spectroscopic analyses of incubations with the various amino acid substrates show a number of long-wavelength absorbing species forming during turnover, tentative assignments are suggested.     

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.     

Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution.

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.     

Purification and characterization of cystathionine gamma-synthase type II from Bacillus sphaericus

Cystathionine gamma-synthase type II, which catalyzes L-cystathionine synthesis from O-acetyl-L-homoserine and L-cysteine was purified from Bacillus sphaericus (IFO 3536) in seven steps. The purified enzyme appeared to be homogeneous by the results of polyacrylamide electrophoresis and ampholyte electrofocusing. The enzyme is a typical pyridoxal-P dependent enzyme, has a molecular mass of 165 kDa and consists of four subunits identical in molecular mass. The enzyme catalyzed the gamma-replacement reaction and the elimination reaction was hardly detected even when a large amount of enzyme was added. In the replacement reaction, O-acetyl-L-homoserine and the following thiol compounds: L and D-cysteine, L and D-homocysteine, sodium sulfide, various alkyl and aryl mercaptans, acted as the most suitable substrate to produce L-cystathionine and the corresponding S-substituted L-homocysteine derivatives.     

The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity.

Cystathionine gamma-synthase catalyses the committed step of de novo methionine biosynthesis in micro-organisms and plants, making the enzyme an attractive target for the design of new antibiotics and herbicides. The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum has been solved by Patterson search techniques using the structure of Escherichia coli cystathionine gamma-synthase. The model was refined at 2.9 A resolution to a crystallographic R -factor of 20.1 % (Rfree25.0 %). The physiological substrates of the enzyme, L-homoserine phosphate and L-cysteine, were modelled into the unliganded structure. These complexes support the proposed ping-pong mechanism for catalysis and illustrate the dissimilar substrate specificities of bacterial and plant cystathionine gamma-synthases on a molecular level. The main difference arises from the binding modes of the distal substrate groups (O -acetyl/succinyl versusO -phosphate). Central in fixing the distal phosphate of the plant CGS substrate is an exposed lysine residue that is strictly conserved in plant cystathionine gamma-synthases whereas bacterial enzymes carry a glycine residue at this position. General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E. coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types.     

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.     

Catalytic action of L-methionine gamma-lyase on selenomethionine and selenols.

We examined the catalytic action of L-methionine gamma-lyase (EC 4.4.1.11) on selenomethionine (2-amino-4-(methylseleno)butyric acid), methaneselenol, l-hexaneselenol, and benzeneselenol. The enzyme catalyzes alpha, gamma-elimination of selenomethionine to yield alpha-letobutyrate, ammonia, and methaneselenol, and also its gamma-replacement reaction with various thiols to produce S-substituted homocysteines. Selenomethionine is an even better substrate than methionine in alpha, gamma-elimination but is less effective in gamma-replacement. In addition, L-methionine gamma-lyase catalyzes gamma-replacement reaction of methionine and its derivatives with selenols to form the corresponding Se-substituted selenohomocysteines, although selenols are less efficient substituent donors than thiols. This is the first proven mechanism for the incorporation of selenium atom into amino acids.     

Cloning, purification and characterisation of cystathionine gamma-synthase from Nicotiana tabacum

Cystathionine gamma-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine gamma-synthase catalyses the pyridoxal 5'-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1+/-3.1 mM and of 0.23+/-0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (Ki = 18 microM, k(inact) = 0.56 min(-1)), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 0.20, 0.30, 0.45, and 0.027 mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine gamma-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine gamma-synthase diffracting to beyond 2.7 A resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 120.0 A, b = 129.5 A, c = 309.8 A, corresponding to two tetramers per asymmetric unit.     

Yeast cystathionine beta-synthase reacts with L-allothreonine, a non-natural substrate, and L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine

Our studies of the reaction mechanism of cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme. The enzyme catalyzes the reaction of L-serine with L-homocysteine to form L-cystathionine through a series of pyridoxal phosphate intermediates. In this work, we explore the substrate specificity of the enzyme by use of substrate analogues combined with kinetic measurements under pre-steady-state conditions and with circular dichroism and fluorescence spectroscopy under steady-state conditions. Our results show that L-allothreonine, but not L-threonine, serves as an effective substrate. L-Allothreonine reacts with the pyridoxal phosphate cofactor to form a stable 3-methyl aminoacrylate intermediate that absorbs maximally at 446 nm. The rapid-scanning stopped-flow results show that the binding of L-allothreonine as the external aldimine is faster than formation of the 3-methyl aminoacrylate intermediate. The 3-methyl aminoacrylate intermediate reacts with L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine, which was characterized by nuclear magnetic resonance spectroscopy. This new amino acid may be a useful analogue of L-cystathionine.     

Cystathionine gamma-lyase of Streptomyces phaeochromogenes. The occurrence of cystathionine gamma-lyase in filamentous bacteria and its purification and characterization

Cystathionine gamma-lyase (EC 4.4.1.1) is widely distributed in actinomycetes, e.g. genera Streptomyces, Micromonospora, Micropolyspora, Mycobacterium, Nocardia, Streptosporangium, and Streptoverticillium. The enzyme was purified from Streptomyces phaeochromogenes (IFO 3105) in nine steps. After the last steps, the enzyme appeared to be homogenous by the criteria of polyacrylamide gel electrophoresis, analytical centrifugation, and double diffusion in agarose. The enzyme crystallized in the apo form with the addition of ammonium sulfate. The enzyme has a molecular weight of about 166,000 and consists of four subunits identical in molecular weight. The enzyme exhibits absorption maxima at 278 and 421 nm and contains 4 mol of pyridoxal 5'-phosphate/mol of enzyme. L-Cystathionine, L-homoserine, DL-lanthionine, L-djenkolic acid, and L-cystine are cleaved as preferred substrates by the Streptomyces enzyme. The alpha, beta-elimination reaction of L-cystathionine is also catalyzed by the enzyme at a ratio of about one-seventh of the alpha, gamma-elimination reaction. Cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-synthase (EC 4.2.99.9) activities were also detected in crude extracts of S. phaeochromogenes, but cystathionine beta-lyase (EC 4.4.1.8) was not. Consequently, the reverse transsulfuration pathway in actinomycetes may be similar to that in yeast and molds.     

Reaction of indole and analogues with amino acid complexes of Escherichia coli tryptophan indole-lyase: detection of a new reaction intermediate by rapid-scanning stopped-flow spectrophotometry

The effects of indole and analogues on the reaction of Escherichia coli tryptophan indole-lyase (tryptophanase) with amino acid substrates and quasisubstrates have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Indole binds rapidly (within the dead time of the stopped-flow instrument) to both the external aldimine and quinonoid complexes with L-alanine, and the absorbance of the quinonoid intermediate decreases in a subsequent slow relaxation. Indoline binds preferentially to the external aldimine complex with L-alanine, while benzimidazole binds selectively to the quinonoid complex of L-alanine. Indole and indoline do not significantly affect the spectrum of the quinonoid intermediates formed in the reaction of the enzyme with S-alkyl-L-cysteines, but benzimidazole causes a rapid decrease in the quinonoid peak at 512 nm and the appearance of a new peak at 345 nm. Benzimidazole also causes a rapid decrease in the quinonoid peak at 505 nm formed in the reaction with L-tryptophan and the appearance of a new absorbance peak at 345 nm. Furthermore, addition of benzimidazole to solutions of enzyme, potassium pyruvate, and ammonium chloride results in the formation of a similar absorption peak at 340 nm. This complex reacts rapidly with indole to form a quinonoid intermediate very similar to that formed from L-tryptophan. This new intermediate is formed faster than catalytic turnover (kcat = 6.8 s-1) and may be an alpha-aminoacrylate intermediate bound as a gem-diamine.     

Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae.

A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.     

Investigation of the open ring form of nicotinamide adenine dinucleotide.

In strong alkali, nicotinamide adenine dinucleotide (NAD+) undergoes a ring opening of the nicotinamide ring. The open form of NAD+, ONAD, has two pKa values at--1.9 and 11.2 and absorbs maximally at 350 nm in its acidic form, at 372 nm in its neutral form, and at 340 nm in its aniomic form. ONAD has the chemical properties expected for a Schiff base of 2-carboxamideglutacondialdehyde (CGDA) and adenosine diphosphate ribosylamine. The decomposition of ONAD has been studied over a wide range of pH. A final product of ONAD hydrolysis is the base fluorescent compound 2-hydroxynicotinaldehyde. In the pH range 10--13, CGDA can be trapped as an intermediate, which absorbs maximally at 345 nm in its anionic form and at 320 nm in its neutral form, pKa = 2.9. The yield of 2-hydroxynicotinaldehyde from ONAD has been estimated as 95% at NaOH concentrations of 5 N and above, and is postulated to result from ring closure of CGDA. The pseudobase hydroxide ring addition adduct of NAD+, psiNAD-OH, is reversibly formed from NAD+ and is the 370-nm precursor of ONAD.     

Conversion of the aminocrotonate intermediate limits the rate of gamma-elimination reaction catalyzed by L-cystathionine gamma-lyase of the yeast Saccharomyces cerevisiae

L-Cystathionine gamma-lyase [EC 4.4.1.1] of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5'-phosphate, up to 2 molecules/subunit. The association constants of the enzyme for the cofactor were estimated to be 3.67 x 10(5) M(-1) and 9.05 x 10(3) M(-1). However, the latter value was too small for the binding to play a catalytic role. Changes in the absorption spectra of the enzyme in gamma-elimination reaction mixtures with various amino acids as substrates were observed at 10 degrees C to elucidate the reaction mechanism of the enzyme. The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-L-homoserine, L-cystathionine, L-homoserine, or O-acetyl-L-homoserine, at rates in this order. The intermediate was consumed at much lower rates than those of formation. The order of the rates of consumption was the same as the order of the formation rates and the order of the gamma-elimination activity of the enzyme with the above-mentioned substrates. These results strongly suggested that the intermediate was essential for gamma-elimination and that the reaction was rate-limited by its conversion into the product alpha-ketobutyrate. L-Cysteine sensitively inhibited the alpha, gamma-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate. The reason for these phenomena is discussed.     

Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies [Lane, A., & Kirschner, K. (1983) Eur. J. Biochem. 129, 561-570], the reaction with alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3). The RSSF data reveal that during tau 1, the internal aldimine, E(PLP), with lambda max = 412 nm (pH 7.8), undergoes rapid conversion to two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process (1/tau 2, 1/tau 3) to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in tau 2 are nearly identical with the spectral changes in tau 3. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with lambda max congruent to 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the alpha proton from the external aldimine. The reaction of L-serine with beta 2 consists of two relaxations (1/tau 1 beta greater than 1/tau 2 beta) and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report [Miles, E. W., Hatanaka, M., & Crawford, I. P. (1968) Biochemistry 7, 2742-2753]. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in tau 2 and tau 3 of the alpha 2 beta 2 reaction is postulated to originate from the existence of two (slowly) interconverting forms of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

A kinetic model of the branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana.

This work proposes a model of the metabolic branch-point between the methionine and threonine biosynthesis pathways in Arabidopsis thaliana which involves kinetic competition for phosphohomoserine between the allosteric enzyme threonine synthase and the two-substrate enzyme cystathionine gamma-synthase. Threonine synthase is activated by S-adenosylmethionine and inhibited by AMP. Cystathionine gamma-synthase condenses phosphohomoserine to cysteine via a ping-pong mechanism. Reactions are irreversible and inhibited by inorganic phosphate. The modelling procedure included an examination of the kinetic links, the determination of the operating conditions in chloroplasts and the establishment of a computer model using the enzyme rate equations. To test the model, the branch-point was reconstituted with purified enzymes. The computer model showed a partial agreement with the in vitro results. The model was subsequently improved and was then found consistent with flux partition in vitro and in vivo. Under near physiological conditions, S-adenosylmethionine, but not AMP, modulates the partition of a steady-state flux of phosphohomoserine. The computer model indicates a high sensitivity of cystathionine flux to enzyme and S-adenosylmethionine concentrations. Cystathionine flux is sensitive to modulation of threonine flux whereas the reverse is not true. The cystathionine gamma-synthase kinetic mechanism favours a low sensitivity of the fluxes to cysteine. Though sensitivity to inorganic phosphate is low, its concentration conditions the dynamics of the system. Threonine synthase and cystathionine gamma-synthase display similar kinetic efficiencies in the metabolic context considered and are first-order for the phosphohomoserine substrate. Under these conditions outflows are coordinated.     

Visualization of PLP-bound intermediates in hemeless variants of human cystathionine beta-synthase: evidence that lysine 119 is a general base

Cystathionine beta-synthase catalyzes the condensation of serine and homocysteine to give cystathionine in a pyridoxal phosphate (PLP)-dependent reaction. The human enzyme contains a single heme per monomer that is bound in an N-terminal 69 amino acid extension that is missing from the otherwise highly homologous yeast enzyme. The heme dominates the UV-visible spectrum and obscures kinetic characterization of the PLP-bound reaction intermediates. In this study, we have engineered a hemeless mutant of human cystathionine beta-synthase by deletion of the N-terminal 69 amino acids. The resulting variant displays approximately 40% of the activity seen with the wild type enzyme, binds stoichiometric amounts of PLP, and permits spectral characterization of PLP-based intermediates. The enzyme as isolated exhibits an absorption maximum at 412nm corresponding to a protonated internal aldimine. Addition of serine shifts the lambdamax to 420nm (assigned as the external aldimine) with a broad shoulder between 450 and 500nm (assigned as the aminoacrylate intermediate). Addition of the product, cystathionine, also leads to formation of an external aldimine (420nm). Homocysteine elicits a red shift (and a decrease in absorption) in the spectrum from 412 to 424nm and an increase in absorption at 330nm, presumably due to formation of a dead-end complex. Mutation of K119, the residue that forms the Schiff base, to alanine results in a approximately 10(3)-fold decrease in activity, which increases approximately 2-fold in the presence of an exogenous base, ethylamine. Spectral shifts (412 --> 420nm) consistent with the formation of external aldimines are observed in the presence of serine or cystathionine, but an aminoacrylate intermediate is not formed at detectable levels. These results are consistent with an additional role for K119 as a general base in the reaction catalyzed by human cystathionine beta-synthase.     

The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine

Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.