Development of polymorphic microsatellite markers for Indian tobacco, Lobelia inflata (Campanulaceae)(1.).

PREMISE OF THE STUDY: Nuclear microsatellite markers were developed for Lobelia inflata (Campanulaceae), an obligately self-fertilizing plant species, for use in the study of temporal fluctuation in allele frequency and of the genetic structure within and among populations. • METHODS AND RESULTS: We developed 28 primer pairs for L. inflata, all of which amplify CT dinucleotide repeats. We evaluated amplification of these loci in 53 L. inflata individuals at three sites in eastern North America and found that 24 loci showed microsatellite polymorphism. We also found that 16 loci amplified successfully in L. cardinalis, and 11 amplified successfully in L. siphilitica. • CONCLUSIONS: These primers will be useful for assessing allelic diversity within and among populations of L. inflata, and show potential for use in congeneric species.

Expression and characterization of the Arabidopsis thaliana 11S globulin family.

The 11S globulins are the principal seed storage proteins in a variety of major crop species, including members of the legume and mustard families. They are targets for protein engineering studies attempting to alter the physicochemical properties of seed protein extracts (e.g. soybean) and to improve the nutritional quality of important agricultural crops. A key factor that has limited the success of this approach to date is insufficient accumulation of the engineered protein variants in vivo due to their improper folding and/or reduced stability, compared to the native protein. We have developed the Arabidopsis thaliana 11S proglobulins as a model system to enable studies exploring the factors underlying structural stability in this family of proteins. Yields of 1.5-4 mg/L were achieved for the three A. thaliana 11S proglobulins expressed in the Origami Escherichia coli cell line in super broth media at 20°C for 16 h and purified via immobilized-metal affinity chromatography. We also demonstrate that differential scanning fluorimetry is an effective and accessible technique to facilitate the screening of variants to enable the successful engineering of 11S seed storage proteins. The relative in vitro stability of the A. thaliana 11S proglobulins (proAtCRU1>proAtCRU3>proAtCRU2) is consistent between chemical and thermal denaturation studies.

A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity.

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4-1.2 pH units, likely due to repositioning of the cofactor and modification of the pKa of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine ß-lyase. The effect of substituting E48, E333 or both residues is the 1.3-3, 26-58 and 124-568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The Km(l-Cth) of E333 substitution variants is increased ~17-fold, while Km(l-OAS) is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (kcat/Km(l-OSHS)=7±2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (kcat/Km(l-Cth)=2100±100) and 260-fold higher than that of l-Hcys (kcat/Km(l-Hcys)=0.027±0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.

Exploration of the six tryptophan residues of Escherichia coli cystathionine ß-lyase as probes of enzyme conformational change.

Cystathionine ß-lyase (CBL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-homocysteine (l-Hcys), pyruvate and ammonia, in the second step of the transsulfuration pathway of bacteria and plants. A series of 17 site-directed variants of Escherichia coli CBL (eCBL) was constructed to probe the contributions of the six tryptophan residues (W131, W188, W230, W276, W300 and W340) to the fluorescence spectrum of eCBL and to assess their mutability and utility as conformational probes. The effects of these Trp→Phe substitutions on kcat and Km(l)(-Cth) are less than 2-fold, with the exception of the 8-fold increase in Km(l)(-Cth) observed for eCBL-W340F. The midpoint of thermal denaturation, as monitored by circular dichroism spectroscopy, is reduced 4.7°C by the W188F substitution while the targeted replacement of the other five tryptophans alter Tm by less than 1.7°C. The fluorescence spectrum of eCBL is dominated by W230 and the contribution of W340, situated in the active site, is minor. The observed 5-fold increase in the 336 nm fluorescence emission of W188 between 0 and 2M urea, suggests a conformational change at the domain interface. Residues W188 and W340, conserved in proteobacterial CBL enzymes, are situated at the core of the domain interface that forms the active-site cleft. The results of this study suggest that W188 is a useful probe of subtle conformational changes at the domain interface and active site.

Exploration of structure-function relationships in Escherichia coli cystathionine γ-synthase and cystathionine ß-lyase via chimeric constructs and site-specific substitutions.

Cystathionine γ-synthase (CGS) and cystathionine ß-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,ß versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80°C, compared to 66°C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.

Identification of cystathionine γ-synthase and threonine synthase from Cicer arietinum and Lens culinaris.

In plants, cystathionine γ-synthase (CGS) and threonine synthase (TS) compete for the branch-point metabolite O-phospho-L-homoserine. These enzymes are potential targets for metabolic engineering studies, aiming to alter the flux through the competing methionine and threonine biosynthetic pathways, with the goal of increasing methionine production. Although CGS and TS have been characterized in the model organisms Escherichia coli and Arabidopsis thaliana, little information is available on these enzymes in other, particularly plant, species. The functional CGS and TS coding sequences from the grain legumes Cicer arietinum (chickpea) and Lens culinaris (lentil) identified in this study share approximately 80% amino acid sequence identity with the corresponding sequences from Glycine max. At least 7 active-site residues of grain legume CGS and TS are conserved in the model bacterial enzymes, including the catalytic base. Putative processing sites that remove the targeting sequence and result in functional TS were identified in the target species.

Exploration of the active site of Escherichia coli cystathionine γ-synthase.

Cystathionine γ-synthase (CGS) catalyzes the condensation of O-succinyl-L-homoserine (L-OSHS) and L-cysteine (L-Cys), to produce L-cystathionine (L-Cth) and succinate, in the first step of the bacterial transsulfuration pathway. In the absence of L-Cys, the enzyme catalyzes the futile α,γ-elimination of L-OSHS, yielding succinate, α-ketobutyrate, and ammonia. A series of 16 site-directed variants of Escherichia coli CGS (eCGS) was constructed to probe the roles of active-site residues D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361. The effects of these substitutions on the catalytic efficiency of the α,γ-elimination reaction range from a reduction of only ∼2-fold for R49K and the E325A,Q variants to 310- and 760-fold for R361K and R48K, respectively. A similar trend is observed for the k(cat) /K(m)(l-OSHS) of the physiological, α,γ-replacement reaction. The results of this study suggest that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in bacterial CGS sequences, tether the distal and α-carboxylate moieties, respectively, of the L-OSHS substrate. In contrast, with the exception of the 13-fold increase observed for R106A, the K(m)(l-Cys) is not markedly affected by the site-directed replacement of the residues investigated. The decrease in k(cat) observed for the S326A variant reflects the role of this residue in tethering the side chain of K198, the catalytic base. Although no structures exist of eCGS bound to active-site ligands, the roles of individual residues is consistent with the structures inhibitor complexes of related enzymes. Substitution of D45, E325, or Y101 enables a minor transamination activity for the substrate L-Ala.

Development of a continuous assay and steady-state characterization of Escherichia coli threonine synthase.

Threonine synthase (TS) catalyzes the hydrolysis of O-phospho-L-homoserine (OPHS) to produce L-threonine (L-Thr) and inorganic phosphate. Here, we report a simplified purification protocol for the OPHS substrate and a continuous, coupled-coupled, spectrophotometric TS assay. The sequential actions of threonine deaminase (TD) and hydroxyisocaproate dehydrogenase (HO-HxoDH) convert the L-Thr product of TS to α-ketobutyrate (α-KB) and then to 2-hydroxybutyrate, respectively, and are monitored as the decrease in absorbance at 340 nm resulting from the concomitant oxidation of ß-nicotinamide adenine dinucleotide (NADH) to NAD(+) by HO-HxoDH. The effect of pH on the activities of Escherichia coli TD and Lactobacillus delbrueckii HO-HxoDH was determined to establish this continuous assay as suitable for steady-state characterization and to facilitate the optimization of coupling enzyme concentrations under different assay conditions to enable studies of TS across phyla. To validate this assay, TS from E. coli was characterized. The kinetic parameters (k(cat)=4s(-1) and K(m)=0.34 mM) and the pH optimum of 8.7, determined using the continuous assay, are consistent with values reported for this enzyme based on the discontinuous malachite green assay. The k(cat)/K(m)(OPHS) versus pH profile of E. coli TS is bell-shaped, and the apparent pK(a) values for the acidic and basic limbs are 7.1 and 10.4, respectively.

Characterization of the side-chain hydroxyl moieties of residues Y56, Y111, Y238, Y338, and S339 as determinants of specificity in E. coli cystathionine ß-lyase.

Cystathionine ß-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of site-directed variants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of the hydroxyl moieties of active-site residues Y56, Y111, Y238, Y338, and S339 as determinants of specificity. The effect of these conservative substitutions on the k(cat)/K(m)(L-Cth) for the α,ß-elimination of L-Cth ranges from a change of only 1.1-fold for Y338F to a reduction of 3 orders of magnitude for the alanine replacement variant of S339. A novel role for residue S339 as a determinant of reaction specificity, via tethering of the catalytic base, K210, is demonstrated. Comparison of the kinetic parameters for L-Cth hydrolysis with those for the inhibition of eCBL by aminoethoxyvinylglycine (AVG) indicates that Y238 interacts with the distal carboxylate group of the substrate. The 22 and 50-fold increases in the K(m)(L-Cth) and K(i)(AVG) resulting from replacement of Y56 with phenylalanine suggest that this residue may interact with the distal amino group of these compounds, although an indirect role in binding is more likely. The near-native k(cat)/K(m)(L-Cth) and pH profile of the eCBL-Y111F variant demonstrate that residue Y111 does not play a role in proton transfer. The understanding of the eCBL active site and of the determinants of substrate and reaction specificity resulting from this work will facilitate the design of inhibitors, as antibacterial therapeutics, and the engineering of enzymes dependent on the catalytically versatile pyridoxal 5'-phosphate cofactor to modify reaction specificity.

The enzymes of the transsulfuration pathways: active-site characterizations.

The diversity of reactions catalyzed by enzymes reliant on pyridoxal 5'-phosphate (PLP) demonstrates the catalytic versatility of this cofactor and the plasticity of the protein scaffolds of the major fold types of PLP-dependent enzymes. The enzymes of the transsulfuration (cystathionine γ-synthase and cystathionine ß-lyase) and reverse transsulfuration (cystathionine ß-synthase and cystathionine γ-lyase) pathways interconvert l-cysteine and l-homocysteine, the immediate precursor of l-methionine, in plants/bacteria and yeast/animals, respectively. These enzymes provide a useful model system for investigation of the mechanisms of substrate and reaction specificity in PLP-dependent enzymes as they catalyze distinct side chain rearrangements of similar amino acid substrates. Exploration of the underlying factors that enable enzymes to control the substrate and reaction specificity of this cofactor will enable the engineering of these properties and the development of therapeutics and antimicrobial compounds. Recent studies probing the role of active-site residues, of the enzymes of the transsulfuration pathways, as determinants of substrate and reaction specificity are the subject of this review. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Residue N84 of yeast cystathionine beta-synthase is a determinant of reaction specificity.

Cystathionine beta-synthase (CBS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the reverse transsulfuration pathway. Residue N84 of yeast CBS (yCBS), predicted to form a hydrogen bond with the hydroxyl moiety of the PLP cofactor, was mutated to alanine, aspartate and histidine. The truncated form of yCBS (ytCBS, residues 1-353) was employed in this study to eliminate any effects of the C-terminal, regulatory domain. The kcat/KmL-Ser of the N84A, N84D and N84H mutants for the beta-replacement reaction is reduced by a factor of 230, 11000 and 640, respectively. Fluorescence resonance energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and N84A mutant, is altered in N84H and absent in N84D. PLP saturation values of 73%, 30% and 67% were observed for the alanine, aspartate and histidine mutants, respectively, compared to 98% for the wild-type enzyme. A marginal beta-elimination activity was detected for N84D (kcat/KmL-Ser=0.23+/-0.02 M(-1) s(-1)) and N84H (kcat/KmL-Ser=0.34+/-0.06 M(-1) s(-1)), in contrast with wild-type ytCBS and the N84A mutant, which do not catalyze this reaction. The ytCBS-N84D enzyme is also inactivated upon incubation with L-serine, via an aminoacrylate-mediated mechanism. These results demonstrate that residue N84 is essential in maintaining the orientation of the pyridine ring of the PLP cofactor and the equilibrium between the open and closed conformations of the active site.

Characterization of site-directed mutants of residues R58, R59, D116, W340 and R372 in the active site of E. coli cystathionine beta-lyase.

Cystathionine beta-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of active-site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k(cat)/K(m) (L-Cth) for the beta-elimination reaction range from a reduction of only 3-fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L-Cth is: R372 >> R58 > W340 approximately R59 > D116. Comparison of the kinetic parameters for L-Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the alpha-carboxylate moiety. The increase in the pK(a) of the acidic limb and decrease in the pK(a) of the basic limb of the k(cat)/K(m) (L-Cth) versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pK(a) of an active-site residue.

Investigation of residues Lys112, Glu136, His138, Gly247, Tyr248, and Asp249 in the active site of yeast cystathionine beta-synthase.

Cystathionine beta-synthase (CBS), the first enzyme of the reverse transsulfuration pathway, catalyzes the pyridoxal 5'-phosphate-dependent condensation of l-serine and l-homocysteine to form l-cystathionine (l-Cth). A model of the l-Cth complex of the truncated form of yeast CBS (ytCBS), comprising the catalytic core, was constructed to identify residues involved in the binding of l-homocysteine and the distal portion of l-Cth. Residue K112 was selected for site-directed mutagenesis based on the results of the in silico docking of l-Cth to the modeled structure of ytCBS. Residues E136, H138, Y248, and D249 of ytCBS were also targeted as they correspond to identical polar residues lining the mouth of the active site in the structure of human CBS. A series of 8 site-directed mutants was constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q > K112L asymptotically equal to K112R >> Y248F > D249A asymptotically equal to H138F > E136A. The beta-replacement activity of G247S, which corresponds to the homocystinuria-associated G307S mutant of human CBS, is undetectable. The Kml-Ser of the K112L and K112R mutants is increased by 50- and 90-fold, respectively, while Kml-Hcys increases by only 2- and 4-fold, respectively. The Kml-Hcys of H138F and Y248F is increased by 8- and 18-fold, respectively. These results indicate that, while the targeted residues are not direct determinants of l-Hcys binding, G307, Y248, and K112 play essential roles in the maintenance of appropriate active-site conformation.

Interconversion of a pair of active-site residues in Escherichia coli cystathionine gamma-synthase, E. coli cystathionine beta-lyase, and Saccharomyces cerevisiae cystathionine gamma-lyase and development of tools for the investigation of their mechanisms and reaction specificity.

Cystathionine gamma-synthase (CGS) and cystathionine beta-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine gamma-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share approximately 30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/KmL-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/KmL-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The DeltametB and DeltametC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

Characterization of the S289A,D mutants of yeast cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue S289 of yeast CBS, predicted to form a hydrogen bond with the pyridine nitrogen of the PLP cofactor, was mutated to alanine and aspartate. The k(cat)/K(m)(l-Ser) of the S289A mutant is reduced by a factor of approximately 800 and the beta-replacement activity of the S289D mutant is undetectable. Fluorescence energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and diminished in the S289A mutant, is absent in S289D. These results demonstrate that residue S289 is essential in maintaining the properties and orientation of the pyridine ring of the PLP cofactor. The reduction in activity of ytCBS-S289A suggests that ytCBS catalyzes the alpha,beta-elimination of l-Ser via an E1cB mechanism.

Kinetic characterization of recombinant human cystathionine beta-synthase purified from E. coli.

Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chromatographic steps as well as proteolytic cleavage to remove the fusion partner. Therefore, a series of five expression constructs, each incorporating a 6-His tag, were developed to enable the efficient purification of hCBS via immobilized metal ion affinity chromatography. Two of the constructs express hCBS in fusion with a protein partner, while the others bear only the affinity tag. The addition of an amino-terminal, 6-His tag, in the absence of a protein fusion partner and in the absence or presence ofa protease-cleavable linker, was found to be sufficient for the purification of soluble hCBS and resulted in enzyme with 86-91% heme saturation and with activity similar to that reported for other hCBS expression constructs. The continuous assay for L-Cth production, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, was employed here for the first time to determine the steady-state kinetic parameters of hCBS, via global analysis, and revealed previously unreported substrate inhibition by L-Hcys (K(i)(L-HCYS) = 2.1 +/- 0.2 mM). The kinetic parameters for the hCBS-catalyzed hydrolysis of L-Cth toL-Ser and L-Hcys were also determined and the k(cat)/K(m)(L-CTH) of this reaction is only approximately 2-fold lower than the k(cat)/K(m)(L-SER) of the physiological, condensation reaction.

The enzymology of cystathionine biosynthesis: strategies for the control of substrate and reaction specificity.

The ability of enzymes to catalyze specific reactions, while excluding others, is central to cellular metabolism. Control of reaction specificity is of particular importance for enzymes that employ catalytically versatile cofactors, of which pyridoxal 5'-phosphate is a prime example. Cystathionine gamma-synthase and cystathionine beta-synthase are the first enzymes in the transsulfuration and reverse transsulfuration pathways, respectively. Each of them occupies branch-point positions in amino acid metabolism and as such are subject to transcriptional and post-translational regulation. Both enzymes catalyze the pyridoxal 5'-phosphate-dependent formation of l-cystathionine; however, their substrate and reaction specificities are distinct. The mechanisms whereby these enzymes control the chemistry of the cofactor are the subject of this review.

Aromatic hydroxamic acids and hydrazides as inhibitors of the peroxidase activity of prostaglandin H2 synthase-2.

The cyclooxygenase activity of the bifunctional enzyme prostaglandin H(2) synthase-2 (PGHS-2) is the target of non-steroidal anti-inflammatory drugs. Inhibition of the peroxidase activity of PGHS has been less studied. Using Soret absorption changes, the binding of aromatic hydroxamic acids to the peroxidase site of PGHS-2 was examined to investigate the structural determinants of inhibition. Typical of mammalian peroxidases, the K(d) for benzhydroxamic acid (42mM) is much greater than that for salicylhydroxamic acid (475microM). Binding of the hydroxamic acid tepoxalin (25microM) resulted in only minor Soret changes. However, tepoxalin is an efficient reducing cosubstrate, indicating that it is an alternative electron donor rather than an inhibitor of the peroxidase activity. Aromatic hydrazides are metabolically activated inhibitors of peroxidases. 2-Naphthoichydrazide (2-NZH) caused the time- and concentration-dependent inhibition of both PGHS-2 peroxidase and cyclooxygenase activities. H(2)O(2) was required for the inactivation of both PGHS-2 activities and indomethacin (which binds at the cyclooxygenase site) did not affect the peroxidase inhibitory potency of 2-NZH. A series of aromatic hydrazides were found to be potent inhibitors of PGHS-2 peroxidase activity with IC(50) values in the 6-100microM range for 13 of the 18 hydrazides examined. Selective inhibition of PGHS-2 over myeloperoxidase and horseradish peroxidase isozyme C was increased by certain ring substitutions. In particular, a chloro group para to the hydrazide moiety increased the PGHS-2 selectivity relative to both myeloperoxidase and horseradish peroxidase isozyme C.

Role of active-site residues Thr81, Ser82, Thr85, Gln157, and Tyr158 in yeast cystathionine beta-synthase catalysis and reaction specificity.

Cystathionine beta-synthase (CBS) effects the condensation of l-serine with l-homocysteine to form l-cystathionine. A series of active-site mutants, T81A, S82A, T85A, Q157A/E/H, and Y158F, was constructed to investigate effects on catalysis and reaction specificity in yeast CBS (yCBS). The effects of these mutations on the k(cat)/K(m)(L-Ser) for the beta-replacement reaction range from a reduction of only 3-fold for Y158F to below detectable levels for the Q157A and Q157E mutants. The order of importance of these residues to the beta-replacement reaction is Gln157 >or= Thr81 > Ser82 > Thr85 approximately Tyr158. All seven of the mutant enzymes catalyze a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH(3) and pyruvate. The ping-pong mechanism of CBS was thus expanded to include the latter reaction for these mutants. This activity is not detectable for wild-type yCBS, suggesting that the mutations result in a shift in the equilibrium between the open and the closed conformations of the active site of yCBS-substrate complexes. The Q157H and Y158F mutants additionally suffer suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme.

Escherichia coli cystathionine gamma-synthase does not obey ping-pong kinetics. Novel continuous assays for the elimination and substitution reactions.

Cystathionine gamma-synthase (CGS) is a pyridoxal phosphate-dependent enzyme that catalyzes a gamma-replacement reaction, in which the succinyl group of an O-succinyl-L-homoserine (L-OSHS) is displaced by the thiol of L-cysteine to form L-cystathionine, in the first step of the bacterial transsulfuration pathway. The mechanism of Escherichia coli CGS (eCGS) is ordered with L-OSHS associating before L-Cys (k(catR)/K(mR)(L-OSHS) = 9.8 x 10(4) M(-1) s(-1), where the subscript R denotes the replacement reaction). The mechanism becomes ping-pong (k(catR)/K(mR)(L-OSHS) = 4.9 x 10(4) M(-1) s(-1)) at L-Cys concentrations lower than K(m)(L-Cys). The enzyme also catalyzes a competing gamma-elimination reaction, in which L-OSHS is hydrolyzed to succinate, NH(3), and alpha-ketobutyrate (k(catE)/K(mE)(L-OSHS) = 1350 +/- 90 M(-1) s(-1), where the subscript E denotes the elimination reaction). The k(cat)/K(m)(L-OSHS) versus pH profile of eCGS is bell-shaped for both reactions. The pH optimum and the pK(a) values for the acidic and basic limbs are 7.4, 6.8 +/- 0.1, and 8.0 +/- 0.1, respectively, for the elimination reaction and 7.8, 7.4 +/- 0.1, and 8.3 +/- 0.1, respectively, for the replacement reaction. The internal aldimine of eCGS remains protonated at pH <10.5, and the alpha-amino group of L-OSHS has a pK(a) of 9.71 +/- 0.01; therefore, neither limb of the k(cat)/K(m)(L-OSHS) versus pH profiles can be assigned to aldimine, or to L-OSHS prototropy. Novel continuous assays for the elimination reaction, employing D-2-hydroxyisocaproate dehydrogenase, and for the substitution reaction, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, are described.