Structural analysis of the substrate recognition mechanism in O-phosphoserine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1

L-Cysteine is synthesized from O-acetyl-L-serine (OAS) and sulfide by O-acetylserine sulfhydrylase (OASS; EC 2.5.1.47) in plants and bacteria. O-phosphoserine sulfhydrylase (OPSS; EC 2.5.1.65) is a novel enzyme from the hyperthermophilic aerobic archaeon Aeropyrum pernix K1 (2003). OPSS can use OAS or O-phospho-L-serine (OPS) to synthesize L-cysteine. To elucidate the mechanism of the substrate specificity of OPSS, we analyzed three-dimensional structures of the active site of the enzyme. The active-site lysine (K127) of OPSS forms an internal Schiff base with pyridoxal 5'-phosphate. Therefore, crystals of the complexes formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with either OPS or OAS were prepared and examined by X-ray diffraction analysis. In contrast to that observed for OASS, no significant difference was seen in the overall structure between the free and complexed forms of OPSS. The side chains of T152, S153, and Q224 interacted with the carboxylate of the substrates, as a previous study has suggested. The side chain of R297 has been proposed to recognize the phosphate group of OPS. Surprisingly, however, the position of R297 was significantly unchanged in the complex of the OPSS K127A mutant with the external Schiff base, allowing enough space for an interaction with OPS. The positively charged environment around the entrance of the active site including S153 and R297 is important for accepting negatively charged substrates such as OPS.     

O-Acetylserine sulfhydrylase from Methanosarcina thermophila

Cysteine is the major source of fixed sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteria and Eucarya domains. Though pathways for cysteine biosynthesis have been established for both of these domains, it is unknown how the Archaea fix sulfur or synthesize cysteine. None of the four archaeal genomes sequenced to date contain open reading frames with identities to either O-acetyl-L-serine sulfhydrylase (OASS) or homocysteine synthase, the only sulfur-fixing enzymes known in nature. We report the purification and characterization of OASS from acetate-grown Methanosarcina thermophila, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5'-phosphate and catalyzed the formation of L-cysteine and acetate from O-acetyl-L-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the Eucarya and Bacteria domains. The purified OASS had a specific activity of 129 micromol of cysteine/min/mg, with a K(m) of 500 +/- 80 microM for sulfide, and exhibited positive cooperativity and substrate inhibition with O-acetyl-L-serine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at 36 kDa, and native gel filtration chromatography indicated a molecular mass of 93 kDa, suggesting that the purified OASS is either a homodimer or a homotrimer. The optimum temperature for activity was between 40 and 60 degrees C, consistent with the optimum growth temperature for M. thermophila. The results of this study provide the first evidence for a sulfur-fixing enzyme in the Archaea domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in the Archaea.     

Alpha,beta-elimination reaction of O-acetylserine sulfhydrylase. Is the pyridine ring required?

O-Acetylserine sulfhydrylase (OASS) catalyzes the elimination of acetate from O-acetyl-L-serine (OAS) followed by addition of bisulfide to give L-cysteine. Site-directed mutagenesis has been used to replace the active site serine, S272, which forms a hydrogen bond to N1 of pyridoxal 5'-phosphate (PLP) with alanine and aspartate. Based on UV-visible spectral and steady-state kinetic studies, both mutant enzymes catalyze the elimination reaction with an efficiency equal to that of the wild-type enzyme. Data are consistent with an anti-E(2) reaction proposed for the elimination reaction.     

Cloning and overexpression of the oah1 gene encoding O-acetyl-L-homoserine sulfhydrylase of Thermus thermophilus HB8 and characterization of the gene product

The oah1 gene of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned, sequenced, and overexpressed in Escherichia coli cells. The gene product having a high O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity was purified to homogeneity, with a recovery of approximately 40% and a purification ratio of 81-fold, both calculated from the cell-homogenate. The protein showed molecular masses of approximately 163000 (for the native form) and 47000 (for the subunit). The isoelectric point was pH 6.0. The optimum temperature and pH for the activity were approximately 70 degrees C and pH 7.8, respectively. The enzyme was also shown to be very stable at high temperature (90% activity remaining at 90 degrees C for 60 min at pH 7.8) and in a wide range of pH (pH 4-12 at room temperature). The absorption spectrum showed a peak at 425 nm, and hydroxylamine hydrochloride (0.1 mM) inhibited approximately 90% of the activity, suggesting formation of a Schiff base with pyridoxal 5'-phosphate. The enzyme showed an apparent K(m) value of 6.8 mM for O-acetyl-L-homoserine, a V(max) value of 165 micromol/min per mg of protein at a fixed sulfide concentration of 5 mM, and also an apparent K(m) value of approximately 1.3 mM for sulfide (with 25 mM acetylhomoserine). L-Methionine (1 mM) inhibited the enzyme activity by 67%. Based on these findings, it was discussed that this enzyme might be inactive under ordinary conditions but might become active as an alternative homocysteine synthase in T. thermophilus HB8, only under such conditions as deficiency in transsulfuration, bringing about a sufficient amount of sulfide available in the cell.     

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.     

Characterization of an inducible phenylserine aldolase from Pseudomonas putida 24-1

An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (Km, 1.3 mM), l-erythro-3-phenylserine (Km, 4.6 mM), l-threonine (Km, 29 mM), and L-allo-threonine (Km, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.     

Discovery and characterization of d-phenylserine deaminase from Arthrobacter sp. TKS1

We discovered a D-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from D-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55°C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50°C. The enzyme acted on the D-forms of β-hydroxy-α-amino acids, such as D-threo-phenylserine (K(m), 19 mM), D-serine (K(m), 5.8 mM), and D-threonine (K(m), 102 mM). As L-threonine, D-allo-threonine, L-allo-threonine, and DL-erythro-phenylserine were inert, the enzyme could distinguish D-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO(4), CuSO(4), BaCl(2), and CoCl(2) and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and DL-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.     

Selenocysteine lyase, a novel enzyme that specifically acts on selenocysteine. Mammalian distribution and purification and properties of pig liver enzyme

We have found a novel enzyme that exclusively decomposes L-selenocysteine into L-alanine and H2Se in various mammalian tissues, and have named it selenocysteine lyase. The enzyme from pig liver has been purified to homogeneity. It has a molecular weight of approximately 85,000, and contains pyridoxal 5'-phosphate as a coenzyme. Its maximum reactivity is at about pH 9.0. Balance studies showed that 1 mol of selenocysteine is converted to equimolar amounts of alanine and H2Se. The following amino acids are insert: L-cysteine, L-serine, L-cysteine sulfinate, selenocysteamine, Se-ethyl-DL-selenocysteine, and L-selenohomocysteine. L-Cysteine (Ki, 1.0 mM) competes with L-selenocysteine (Km, 0.83 mM) to inhibit the enzyme reaction. The enzyme is the first proven enzyme that specifically acts on selenium compounds.     

pH dependence of the absorbance and 31P NMR spectra of O-acetylserine sulfhydrylase in the absence and presence of O-acetyl-L-serine.

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme which catalyzes the final step in the biosynthesis of L-cysteine in Salmonella, viz., the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. UV-visible spectra of OASS exhibit absorbance maxima at 280 and 412 nm with pH-independent extinction coefficients over the range 5.5-10.8. Addition of OAS to enzyme results in a shift in the absorbance maximum from 412 to 470 nm, indicating the formation of an alpha-aminoacrylate Schiff base intermediate [Cook, P. F., & Wedding, R. T. (1976) J. Biol. Chem. 251, 2023]. The spectrum of the intermediate is also pH independent from 5.5 to 9.2. The observed changes in absorbance at 470 nm at different concentrations of OAS were used to calculate a Kd of 3 microM for OAS at pH 6.9. As the pH decreases, the Kd increases an order of magnitude per pH unit. The 31P NMR signal of the bound PLP has a pH-independent chemical shift of 5.2 ppm in the presence and absence of OAS. These results indicate that the phosphate group is present as the dianion possibly salt-bridged to positively charged groups of the protein. In agreement with this, the resonance at 5.2 ppm has a line width of 20.5 Hz, suggesting that the cofactor is tightly bound to the protein. The sulfhydrylase was also shown to catalyze an OAS deacetylase activity in which OAS is degraded to pyruvate, ammonia, and acetate. The activity was detected by a time-dependent disappearance of the 470-nm absorbance reflecting the alpha-aminoacrylate intermediate. The rate of disappearance of the intermediate was measured at pH values from 7 to 9.5 using equal concentrations of OAS and OASS. The rate constant for disappearance of the intermediate decreases below a pK of 8.1 +/- 0.1, reflecting the deprotonation of the active-site lysine that originally formed the Schiff base with PLP in free enzyme. A possible mechanism for the deacetylase activity is presented where the lysine displaces alpha-aminoacrylate which decomposes to pyruvate and ammonia.     

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5''-phosphate.

1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.     

Structure and mechanism of O-acetylserine sulfhydrylase

The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a beta-replacement reaction in which the beta-acetoxy group of O-acetyl-L-serine (OAS) is replaced by bisulfide to give L-cysteine and acetate. The kinetic mechanism of OASS is ping-pong with a stable alpha-aminoacrylate intermediate. The enzyme is a homodimer with one pyridoxal 5'-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers. All of the active site residues are contributed by a single subunit. The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain. The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP. The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cbeta-O bond cleavage than for Calpha-H bond cleavage.     

Purification and characterization of pyridoxine 5'-phosphate phosphatase from Sinorhizobium meliloti

Here we report the purification and biochemical characterization of a pyridoxine 5'-phosphate phosphatase involved in the biosynthesis of pyridoxine in Sinorhizobium meliloti. The phosphatase was localized in the cytoplasm and purified to electrophoretic homogeneity by a combination of EDTA/lysozyme treatment and five chromatography steps. Gel-filtration chromatography with Sephacryl S-200 and SDS/PAGE demonstrated that the protein was a monomer with a molecular size of approximately 29 kDa. The protein required divalent metal ions for pyridoxine 5'-phosphate phosphatase activity, and specifically catalyzed the removal of Pi from pyridoxine and pyridoxal 5'-phosphates at physiological pH (about 7.5). It was inactive on pyridoxamine 5'-phosphate and other physiologically important phosphorylated compounds. The enzyme had the same Michaelis constant (K(m)) of 385 muM for pyridoxine and pyridoxal 5'-phosphates, but its specific constant [maximum velocity (V(max))/K(m)] was nearly 2.5 times higher for the former than for the latter.     

Purification and characterization of human hepatic cysteine-conjugate beta-lyase.

Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5'-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.     

Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme

Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.     

Biochemical characterisation of the enzyme responsible for "activated L-serine sulphydrase" activity in nematodes.

The biochemical properties of the enzyme responsible for nematode "activated L-serine sulphydrase" activity (L-cysteine + R-SH----cysteine thioether + H2S) have been investigated using primarily the gastro-intestinal nematodes Nippostrongylus brasiliensis and Haemonchus contortus. The activated L-serine sulphydrase enzyme was found to be cytosolic in origin and exhibited maximal activity at pH 9.0. Enzyme activity was widely distributed amongst the major tissues of adult female Ascaris suum but was particularly abundant in longitudinal muscle. The enzyme appeared to have a rigid specificity for L-cysteine as the primary thiol substrate, but was capable of utilising a number of sulphur amino acids (and derivatives) and nonphysiological thiols as second substrates. The best second thiol substrates were nonphysiological, hydroxyl-containing thiols that showed some structural similarity to the standard second substrate, 2-mercaptoethanol. Kinetic analyses revealed that the enzyme operates by a sequential catalytic mechanism, and the absolute Michaelis constants were: KL-cysteine = 0.21 +/- 0.02 mM and K2-mercaptoethanol = 5.58 +/- 0.59 mM. The enzyme was relatively insensitive to inhibition by a variety of substrate analogues and known inhibitors of pyridoxal 5'-phosphate dependent enzymes, whilst plant phenols caused significant levels of inhibition. The most potent inhibitors discovered were the anthelmintics bithionol, dichlorophene and hexachlorophene. Further characterisation revealed that hexachlorophene was a parabolic competitive inhibitor of the activated L-serine sulphydrase enzyme.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

4-Aminobutyrate:2-oxoglutarate aminotransferase from Candida. Purification and properties

An enzyme which catalyzes the transamination of 4-aminobutyrate with 2-oxoglutarate was purified 588-fold to homogeneity from Candida guilliermondii var. membranaefaciens, grown with 4-aminobutyrate as sole source of nitrogen. An apparent relative molecular mass of 107,000 was estimated by gel filtration. The enzyme was found to be a dimer made up of two subunits identical in molecular mass (Mr 55,000). The enzyme has a maximum activity in the pH range 7.8-8.0 and a temperature optimum of 45 degrees C. 2-Oxoglutarate protects the enzyme from heat inactivation better than pyridoxal 5'-phosphate. The absorption spectrum of the enzyme exhibits two maxima at 412 nm and 330 nm. The purified enzyme catalyzes the transamination of omega-amino acids; 4-aminobutyrate is the best amino donor and low activity is observed with beta-alanine. The Michaelis constants are 1.5 mM for 2-oxoglutarate and 2.3 mM for 4-aminobutyrate. Several amino acids, such as alpha,beta-alanine and 2-aminobutyrate, are inhibitors (Ki = 38.7 mM, Ki = 35.5 mM and Ki = 33.2 mM respectively). Propionic and butyric acids are also inhibitors (Ki = 3 mM and Ki = 2 mM).     

Bovine brain low Mr acid phosphatase: purification and properties

Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.     

Purification and characterization of a novel enzyme, L-threo-3-hydroxyaspartate dehydratase, from Pseudomonas sp. T62

L-threo-3-Hydroxyaspartate dehydratase (L-threo-3-hydroxyaspartate hydro-lyase), which exhibited specificity for L-threo-3-hydroxyaspartate (K(m)=0.74 mM, V(max)=37.5 micromol min(-1) (mg protein)(-1)) but not for D-threo or D, L-erythro-3-hydroxyaspartate, was purified from a cell-free extract of Pseudomonas sp. T62. The activity of the enzyme was inhibited by hydroxylamine and EDTA, which suggests that pyridoxal 5'-phosphate and divalent cations participate in the enzyme reaction. The NH(2)-terminal amino acid sequence showed significant similarity to the Saccharomyces cerevisiae YKL218c gene product, a hypothetical threonine dehydratase. However, the purified enzyme showed no threonine dehydratase activity.     

The equilibrium position of the reaction of bovine liver glutamate dehydrogenase with pyridoxal5''-phosphate. A demonstration that covalent modification with this reagent completely abolishes catalytic activity.

1. The activity of bovine liver glutamate dehydrogenase incubated with pyridoxal 5'-phosphate declined to a steady value reached within 30--60 min. The residual activity depended on the concentration of modifier up to about 5 mM. Above this concentration, however, no further inactivation was produced. The minimum activity obtainable in such incubations was 6--7% of the initial value. 2. Km values of the modified enzyme were unaltered, whereas Vmax. was decreased. 3. Activity was fully regained on dialysis against 0.1 M-potassium phosphate buffer. 4. Reduction with borohydride rendered the inactivation permanent but did not alter its extent. 5. Enzyme permanently inactivated in this way to the extent of 90% and dialysed was re-treated with pyridoxal 5'-phosphate. In this second cycle activity declined from 10 to 1% of the original activity. 6. This strongly suggests that the failure to achieve complete inactivation in a single cycle reflects a reversible equilibrium between inactive Schiff base, i.e. covalently modified enzyme, and a non-covalent complex. 7. The re-inactivation reaction occurring on dilution was demonstrated directly and a first-order rate constant obtained (0.048 min-1). This, in conjunction with an estimate of the forward rate constant for Schiff-base formation, obtained by approximate pseudo-first-order analysis of inactivation at varied modifier concentrations, gives a predicted minimum activity very close to that actually obtained in a single cycle of treatment. 8. The dissociation constant of the non-covalent complex is given by two methods as 0.90 and 1.59mM. 9. The results indicate that covalent modification with pyridoxal 5'-phosphate completely abolishes the activity of glutamate dehydrogenase.