The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death

Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfide-bonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys(149), we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in beta-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys(149). Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.     

An MCD spectroscopic study of the molybdenum active site in sulfite oxidase: insight into the role of coordinated cysteine

Temperature-dependent magnetic circular dichroism (MCD) spectroscopy has been used for the first time to probe the electronic structure of the Mo active site in sulfite oxidase (SO). The enzyme was poised in the catalytically relevant [Mo(V):Fe(II)] state by anaerobic reduction of the enzyme with the natural substrate, sulfite, in the absence of the physiological oxidant cytochrome c. The [Mo(V):Fe(II)] state is of particular importance, as it is proposed to be a catalytic intermediate in the oxidative half reaction, where SO is reoxidized to the resting [Mo(VI):Fe(III)] state by two sequential one-electron transfers to cytochrome c. The MCD spectrum of the enzyme shows no charge transfer transitions below 17 000 cm⁻¹. This has been interpreted to result from (1) a severe reduction in ene-1,2-dithiolate sulfur in-plane and out-of-plane p orbital mixing, (2) a decrease in the dithiolate sulfur out-of-plane p-Mo d_xy orbital overlap, and (3) an orthogonal orientation between the vertical cysteine sulfur p (perpendicular to the Mo–S_cys σ-bond) and Mo d_xy orbitals. The spectroscopically determined cysteine sulfur p-Mo d_xy bonding scheme in the [Mo(V):Fe(II)] state is consistent with the crystallographically determined O–Mo–S_cys–C dihedral angle of 90° and precludes a covalent interaction between the vertical cysteine sulfur p orbital and Mo d_xy, effectively decoupling the cysteine from an effective through-bond electron transfer pathway. We have tentatively assigned a 22 250 cm⁻¹ positive C-term feature in the MCD as the cysteine S(σ)→Mo d_xy charge transfer that becomes allowed by a combination of configuration interaction and low-symmetry; however, the orbital overlap is anticipated to be quite small due to the near orthogonality of these orbitals. Therefore, we propose that the primary role of the coordinated cysteine is to decrease the effective nuclear charge on Mo by charge donation to the metal, statically poising the active site at more negative reduction potentials during electron transfer (ET) regeneration. Finally, the results of this study are consistent with the pyranopterin ene-1,2-dithiolate acting to couple the Mo site into efficient superexchange pathways for ET regeneration following oxygen atom transfer to the substrate.     

Molecular basis of enzyme inactivation by an endogenous electrophile 4-hydroxy-2-nonenal: identification of modification sites in glyceraldehyde-3-phosphate dehydrogenase

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived reactive aldehyde, is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It has been suggested that HNE exerts an inhibitory effect on the enzyme due to the modification of the cysteine residue (Cys-149) at the catalytic site generating the HNE-cysteine Michael addition-type adduct [Uchida, K., and Stadtman, E. R. (1993) J. Biol. Chem. 268, 6388-6393]. In the study presented here, to elucidate the mechanism for the inactivation of GAPDH by HNE, we attempted to identify the modification sites of the enzyme by monitoring the formation of the HNE Michael adducts by mass spectrometric methods. Incubation of GAPDH (1 mg/mL) with 1 mM HNE in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C resulted in a time-dependent loss of enzyme activity, which was associated with the covalent binding of HNE to the enzyme. To identify the site of modification of GAPDH by HNE, both the HNE-pretreated and untreated GAPDH were digested with trypsin and V8 protease, and the resulting peptides were subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS). This technique identified five peptides, which contained the HNE adducts at His-164, Cys-244, Cys-281, His-327, and Lys-331 and revealed that both His-164 and Cys-281 were very rapidly modified at 5 min, followed by Cys-244 at 15 min and His-327 and Lys-331 at 30 min. These observations and the observation that the HNE modification of the catalytic center, Cys-149, was not observed suggest that the HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the selective modification of amino acids primarily located in the surface of the GAPDH molecule.     

Lipolytic activity in submerged cultures of Issatchenkia orientalis

The ability of the yeast Issatchenkia orientalis CECT 10688 to secrete lipolytic activity in submerged culture was investigated. The yeast was grown in a complex medium supplemented with a fixed concentration of several lipidic compounds (triglycerides, fatty acids). Maximum enzyme activity around 70–80 U cm⁻³ was produced in cultures supplemented with tributyrin. An optimum tributyrin concentration of 10 g dm⁻³ was selected. Several surfactants were added to the cultures, but no significant increase in activity was detected. Finally, the effect of the type of carbon source on lipolytic enzyme production was studied. The best results were obtained with glucose and fructose (60–80 U cm⁻³), while rather low enzyme activity was found in cultures grown on lactose and maltose (about 20 U cm⁻³).     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology, lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Myeloperoxidase-catalyzed chlorination: the quest for the active species

Myeloperoxidase (MPO) is a dominating enzyme of circulating polymorphonuclear neutrophils that catalyzes the two-electron oxidation of chloride, thereby producing the strong halogenating agent hypochlorous acid (ClO⁻/HOCl). In absence of MPO the tripeptide Pro-Gly-Gly reacts with HOCl faster than the amino acid taurine (2-aminoethanesulfonic acid, Tau), while the MPO-mediated chlorination shows reverse order. A comparative study of the enzymatic oxidation of both substrates at pH 4.0–6.0, varying H₂O₂ concentration is presented. Initial and equilibrium rates studies have been carried on, reaction rates in the latter being slower due to the chemical equilibrium between MPO-I and MPO-II–HO₂. A maximum of chlorination rate is observed for Pro-Gly-Gly and Tau when [H₂O₂] ≈ 0.3–0.7 mM and pH ≈ 4.5–5.0. Several mechanistic possibilities are considered, the proposed one implies that chlorination takes place via two pathways. One, for bulkier substrates, involves chlorination by free HOCl outside the heme cavity; ClO⁻ is released from the active center, diffuses away the heme cavity, and undergoes protonation to HOCl. The other implies the existence of compound I–Cl⁻ complex (MPO-I–Cl), capable of chlorinating smaller substrates in the heme pocket. Electronic structure calculations show the size of Pro-Gly-Gly comparable to the available gap in the substrate channel, this tripeptide being unable to reach the active site, and its chlorination is only possible by free HOCl outside the enzyme.     

ADP binding and ATP synthesis by reconstituted H-ATPase from chloroplasts

The H⁺-ATPase from chloroplasts, CF₀F₁, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH_in = 5.0, pH_out = 8.5) and a K⁺/valinomycin diffusion potential, Δφ (K⁺_in = 0.6 mM, K⁺_out = 60 mM). A rate of 250 s⁻¹ was observed with the reduced enzyme (85 s⁻¹ in the absence of Δφ). A rate of 50 s⁻¹ was observed with the oxidized enzyme under the same conditions (15 s⁻¹ in the absence of Δφ). The reconstituted enzyme contained 2 ATP_bound per CF₀F₁ and 1 ADP_bound per CF₀F₁. Upon energisation the enzyme was activated and 0.9 ADP per CF₀F₁, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10⁵ M⁻¹·s⁻¹ under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.     

Mechanisms of Nitrosylation and Denitrosylation of Cytoplasmic Glyceraldehyde-3-phosphate Dehydrogenase from Arabidopsis thaliana

Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Purification and characterization of a nitrilase from Fusarium solani O1

An intracellular nitrilase was purified from a Fusarium solani O1 culture, in which the enzyme (up to 3000 U L⁻¹) was induced by 2-cyanopyridine. SDS-PAGE revealed one major band corresponding to a molecular weight of approximately 40 kDa. Peptide mass fingerprinting suggested a high similarity of the protein with the putative nitrilase from Gibberella moniliformis. Electron microscopy revealed that the enzyme molecules associated into extended rods. The enzyme showed high specific activities towards benzonitrile (156 U mg⁻¹) and 4-cyanopyridine (203 U mg⁻¹). Other aromatic nitriles (3-chlorobenzonitrile, 3-hydroxybenzonitrile) also served as good substrates for the enzyme. The rates of hydrolysis of aliphatic nitriles (methacrylonitrile, propionitrile, butyronitrile, valeronitrile) were 14–26% of that of benzonitrile. The nitrilase was active within pH 5–10 and at up to 50 °C with optima at pH 8.0 and 40–45 °C. Its activity was strongly inhibited by Hg²⁺ and Ag⁺ ions. More than half of the enzyme activity was preserved at up to 50% of n-hexane or n-heptane or at up to 15% of xylene or ethanol. Operational stability of the enzyme was examined by the conversion of 45 mM 4-cyanopyridine in a continuous and stirred ultrafiltration-membrane reactor. The nitrilase half-life was 277 and 10.5 h at 35 and 45 °C, respectively.     

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor

Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na⁺-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B₂ receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na⁺-ATPase activity was evaluated. Preincubation of Na⁺-ATPase with 10⁻⁹ M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg⁻¹ min⁻¹, corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10⁻¹⁴–10⁻⁸ M), reaching maximal inhibitory effect at 10⁻⁹ M. Des-Arg⁹ bradykinin (DABK), an agonist of B₁ receptor, at the concentrations of 10⁻⁹–10⁻⁷ M, does not mimic the BK inhibitory effect, and des-Arg⁹-[Leu⁸]-BK (DALBK), a B₁ receptor antagonist, at the concentrations of 10⁻¹⁰–10⁻⁷ M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B₂ receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10⁻⁷ M. Taken together, these data indicate that stimulation of B₂ receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na⁺-ATPase activity.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Novel inhibitors of glyceraldehyde-3-phosphate dehydrogenase: Covalent modification of NAD-binding site by aromatic thiols

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) is a glycolytic enzyme catalyzing the formation of 1,3-diphosphoglycerate from glyceraldehyde-3-phosphate and inorganic phosphate. In cooperation with E3 ubiquitin-kinase Siah1, GAPDH directly participates in the apoptotic death of neurons in Parkinson’s disease. Potential GAPDH inhibitors were screened in silico, and three compounds with high affinity to the NAD-binding site and theoretically capable of forming a disulfide bond with amino acid residue Cys149 were found among cysteine and glutathione derivatives. The inhibitory effect of these compounds was tested on GAPDH from rabbit muscles using isothermal calorimetry and kinetic methods. As a result of experimental screening, we selected two compounds that inhibit GAPDH by forming disulfide bonds with the Cys149 residue in the enzyme active site. Since Cys149 is the key residue not only for the catalyzed reaction, but also for interaction with Siah1, the compounds can be assumed to inhibit the formation of the proapoptotic complex GAPDH-Siah1 and therefore have potential effect against Parkinson’s disease.     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).     

Coenzyme production using immobilized enzymes. I. Preparation, characterization, and laboratory-scale application of an immobilized NAD kinase

NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, EC2.7.1.23) isolated from chicken liver was immobilized on a silica-based support possessing aldehyde functional groups. The highest catalytic activity achieved was 16 U g⁻¹ solid. The optimal pH for the catalytic activity of the immobilized NAD⁺ kinase was pH 7.1–7.3. The apparent optimum temperature for the immobilized enzyme was about 5°C higher than that of the soluble enzyme. There were no significant differences in the K_{m app} values. The immobilization improved the conformational stability of the enzyme. In preliminary experiments, a 95% conversion of NAD⁺ to NADP⁺ was achieved with use of the immobilized NAD⁺ kinase, which preserved its starting activity practically unchanged up to 36 days.     

Chloroplast inorganic pyrophosphatase

An alkaline, Mg²⁺-dependent inorganic pyrophosphatase has been isolated from previously isolated spinach chloroplast. The activity of the enzyme was increased 100-fold, with a 42% yield, upon purification from the total soluble chloroplast enzymes. The pH optimum for the enzyme shifts from 9.0 at 5 mM Mg²⁺ to 7.0 at 40 mM Mg²⁺. The substrate for the reaction appears to be magnesium pyrophosphate, and anionic pyrophosphate is an effective inhibitor. There seems to be also an activating effect of Mg²⁺ on the enzyme at pH 7. No other cation substitutes for Mg²⁺ in activating the hydrolysis of pyrophosphate. Among anions tested, only F⁻ caused severe inhibition. The enzyme is inactive towards fructose 1,6-diphosphate, thiamine pyrophosphate, ATP, and ADP. The possibility that this enzyme is subject to metabolic regulation is discussed in relation to an indicated role of pyrophosphate in the regulation of photosynthetic carbon reduction.