The soluble [NiFe]-hydrogenase from<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> contains four cyanides in its active site,one of which is responsible for the insensitivity towards oxygen

Infrared spectra of ¹⁵N-enriched preparations of the soluble cytoplasmic NAD⁺-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)₂(CN)Ni(-RS)₂Fe(CN)₃(CO) centre (R=Cys) and that H₂ activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.Abbreviations BV benzyl viologen - DCIP 2,6-dichlorophenol-indophenol - EXAFS extended X-ray absorption fine structure - FTIR Fourier transform infrared - MV methyl viologen - SH soluble NAD⁺-reducing hydrogenase - XAS X-ray absorption spectroscopy     

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> and some EPR and IR properties of the hydrogenase module

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M _r = 52 kDa) and a small (M _r = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe–S clusters, three [2Fe–2S] clusters and five [4Fe–4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe–S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H₂-dependent reduction of NAD⁺. Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni–Fe active site and a [4Fe–4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni_a–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.     

FAD-binding site and NADP reactivity in human renalase: a new enzyme involved in blood pressure regulation

Renalase is a recently discovered flavoprotein that regulates blood pressure, regulates sodium and phosphate excretion, and displays cardioprotectant action through a mechanism that is barely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, via either O₂-dependent or NADH-dependent mechanisms. Here we report the renalase crystal structure at 2.5 Å resolution together with new data on its interaction with nicotinamide dinucleotides. Renalase adopts the p-hydroxybenzoate hydroxylase fold topology, comprising a Rossmann-fold-based flavin adenine dinucleotide (FAD)-binding domain and a putative substrate-binding domain, the latter of which contains a five-stranded anti-parallel β-sheet. A large cavity (228 Å³), facing the flavin ring, presumably represents the active site. Compared to monoamine oxidase or polyamine oxidase, the renalase active site is fully solvent exposed and lacks an ‘aromatic cage’ for binding the substrate amino group. Renalase has an extremely low diaphorase activity, displaying lower k_cat but higher k_cat/K_m for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NADPH under anaerobiosis, and binds NAD⁺ or NADP⁺ with K_d values of ca 2 mM. The absence of a recognizable NADP-binding site in the protein structure and its poor affinity for, and poor reactivity towards, NADH and NADPH suggest that these are not physiological ligands of renalase. Although our study does not answer the question on the catalytic activity of renalase, it provides a firm framework for testing hypotheses on the molecular mechanism of its action.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Apurinic acid endonuclease implicated in DNA breakage in Escherichia coli subjected to mild heat.

Nicotinamide nucleotide transhydrogenase from beef heart mitochondria was purified to homogeneity and characterized. The enzyme is devoid of other respiratory chain activities as well as flavin. Reduction of NAD⁺ by NADPH catalyzed by reconstituted transhydrogenase generates an uncoupler-sensitive uptake of lipophilic anions, whereas the rate of reduction of NAD⁺ by NADPH is enhanced about 13 fold by uncouplers. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate reveales that the protein consists of a single polypeptide of a molecular weight of 97,000.     

A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, <Emphasis Type="Italic">Flavobacterium frigidimaris</Emphasis> KUC-1

An NAD⁺-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD⁺ as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD⁺. F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85°C and the most active at 70°C. The half-life time and k _cat value at 60°C were calculated to be 50 min and 27,400 min⁻¹, respectively. The enzyme also shows high catalytic efficiency at low temperatures (0–20°C) (k _cat/K _m at 10°C; 12,600 mM⁻¹ min⁻¹) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.     

l-Myoinositol-1-phosphate synthase from Neurospora crassa: Purification to homogeneity and partial characterization

The purification procedure of milligram quantities of stable myoinositol-1-phosphate synthase (EC 5.5.1.4) from Neurospora crassa is reported. The procedure includes: (a) (NH₄)₂SO₄ and protamine sulfate precipitations, (b) gel filtration in Ultrogel AcA-34 (LKB), (c) DEAE-cellulose chromatography, (d) AH-Sepharose 4B chromatography, and (e) calcium phosphate gel chromatography. The enzyme is considered pure according to the following criteria: (a) gel filtration, (b) sucrose density gradient centrifugation, (c) polyacrylamide gel electrophoresis, and (d) isoelectric focusing technique. The molecular weight estimated by gel filtration chromatography and sucrose density gradient centrifugation is 345,000. The subunit molecular weight is 59,000. The active enzyme seems to posses an hexameric structure. The isoelectric point estimated for the pure enzyme is 5.2. The enzyme was optimally stimulated by 10 mm (NH₄)₂SO₄ and by 50 mm KCl, while NaCl had a minor inhibitory effect at higher concentrations. The divalent cations Mg²⁺ and Mn²⁺ were inhibitory only at nonphysiological concentrations. The enzymatic activity after the salt fractionation steps was about 33% NAD⁺ independent; but with purification the resulting homogeneous enzyme showed less than 5% NAD⁺-independent activity.     

Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD⁺-dependent with a thermal optimum for activity at 55–60°C and an apparent midpoint melting temperature (T _m) of 70°C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD⁺-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD⁺-IDHs with high sequence identity (48–57%) to McIDH were however identified in a variety of bacteria showing that NAD⁺-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD⁺-IDH and the subfamily III of IDH was redefined to include bacterial NAD⁺- and NADP⁺-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD⁺-IDH are derived from the McIDH-like IDHs.     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.     

Purification and characterization of alcohol oxidase from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> isolated as a formaldehyde-resistant fungus

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.     

Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

Glutamate dehydrogenase of lupin nodules: Purification and properties

Glutamate dehydrogenase, GDH (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD⁺ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD⁺. When NAD⁺ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD⁺.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K _m value. Starch-binding assays showed that the K _d and B _max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay

An enzyme assay was developed to determine the activities of methyl chloride dehalogenase and O-demethylase of the homoacetogen strain MC. The formation of methyl tetrahydrofolate from tetrahydrofolate and methyl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate mediated by methylene tetrahydrofolate reductase purified from Peptostreptococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organism. To drive the endergonic methyl tetrahydrofolate oxidation with NAD⁺ as an electron acceptor, the NADH formed in this reaction was reoxidized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofolate dehydrogenase reaction was followed photometrically at 350 nm; ε₃₅₀ was about 29.5 mM^–1cm^–1 (pH 6.5). Using the coupled enzyme assay, the cofactor requirements, the apparent kinetic parameters, the pH and temperature optima of both enzymes, and the effect of inhibitors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate severely inhibited both enzyme activities in the absence of ATP. The coupled enzyme assay described allows purification and characterization of methyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate. Received: 2 August 1995 / Accepted: 28 September 1995     

Oxidation of excited-state NADH and NAD dimer in aqueous medium involvement of O2− as a mediator in the presence of oxygen

It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD⁺ (or NADP⁺). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H₂O₂ at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD⁺ is about 75%. Under analogous irradiation conditions, the dimers (NAD)₂ and (NADP)₂ undergo disproportionation to NAD⁺ and NADP⁺, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)₂.     

Activation of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> inorganic pyrophosphatase and the importance of Cys16 in thermostability,enzyme activation and quaternary structure

The inorganic pyrophosphatase from the human pathogen Helicobacter pylori (HpPPase) is a family I PPase. It is a homohexamer consisting of identical 20-kDa subunits. Hydrolysis of inorganic pyrophosphate (PP_i) by HpPPase relied on the presence of magnesium and followed Michaelis–Menten kinetics, with k _cat being 344 s⁻¹ and K _m being 83 μM at pH 8.0, which was the optimal pH for catalysis. HpPPase was activated by both thiol and non-thiol reductants, distinct from the previously suggested inactivation/reactivation process involving formation and breakage of disulfide bonds. Substitution of Cys16 of HpPPase, which was neither located at the active site nor evolutionarily conserved, resulted in a loss of 50% activity and a reduction in sensitivity to reductants and oxidized glutathione. In addition, the C16S replacement caused a considerable disruption in thermostability, which exceeded that resulted from active-site mutations such as Y140F HpPPase and those of Escherichia coli. Although Cys16 was not located at the subunit interface of the hexameric HpPPase, sedimentation analysis results suggested that the C16S substitution destabilized HpPPase through impairing trimer–trimer interactions. This study provided the first evidences that the single cysteine residue of HpPPase was involved in enzyme activation, thermostability, and stabilization of quaternary structure. Mon-Juan Lee and Haimei Huang contributed equally to this work.     

Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min^–1 (mg protein)^–1. The dismutase obeyed Michaelis-Menten kinetics. The V _max and K _m calculated for chlorite were 2,200 U (mg protein)^–1 and 170 μM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30° C. Received: 9 April 1996 / Accepted: 12 August 1996