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PubMed1992

Differential effects of cyclo-oxygenase and thromboxane synthetase inhibition on ventilation-perfusion relationships in acid aspiration-induced acute lung injury.

Cyclo-oxygenase metabolites are important regulators of pulmonary vascular and airway tone and may act to regulate ventilation-perfusion (VA/Q) relationships. Hypoxemia that follows aspiration of gastric acid is associated with increased venous admixture, and plasma levels of thromboxane (TX) B2 and 6-keto-PGF2 alpha are increased after experimental acid-induced acute lung injury. The present study was designed to determine the effects of cyclo-oxygenase metabolites on VA/Q relationships in canine acid aspiration. Eighteen anesthetized dogs received 0.2 mL/kg 0.1 N HCl intratracheally; six were pretreated with ibuprofen (IBU), a cyclo-oxygenase inhibitor, 12.5 mg/kg IV, and six other dogs received OKY-046 (OKY), a TX synthetase inhibitor, 0.5 mg/kg IV. The remaining six animals (ACID) served as controls. Continuous distributions of ventilation and perfusion were evaluated with the multiple inert gas elimination technique. Within 30 minutes, acid injury resulted in significant (p < 0.05) decreases in PaO2 from baseline values by 44.7 +/- 5.4 and 47.6 +/- 4.8 mm Hg in the ACID and OKY groups, respectively. Although decreased, the change in PaO2 of 21.0 +/- 4.8 mm Hg in IBU animals was significantly (p < 0.05) attenuated in comparison with the other groups. Ibuprofen increased pulmonary vascular resistance, attenuated perfusion to shunt and low VA/Q areas, and reduced ventilation to unperfused areas for the first 2 hours after acid injury (all p < 0.05), whereas OKY exacerbated hypoxemia and VA/Q inequality.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling.

The 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures. Recently, it has been discovered that they act as sensors in the hypoxic response in humans and other animals. Substrate oxidation is coupled to conversion of 2OG to succinate and carbon dioxide. Kinetic, spectroscopic and structural studies are consistent with a consensus mechanism in which ordered binding of (co)substrates enables control of reactive intermediates. Binding of the substrate to the active site triggers the enzyme for ligation of dioxygen to the metal. Oxidative decarboxylation of 2OG then generates the ferryl species thought to mediate substrate oxidation. Structural studies reveal a conserved double-stranded beta-helix core responsible for binding the iron, via a 2His-1carboxylate motif and the 2OG side chain. The rigidity of this core contrasts with the conformational flexibility of surrounding regions that are involved in binding the substrate. Here we discuss the roles of 2OG oxygenases in terms of the generic structural and mechanistic features that render the 2OG oxygenases suited for their functions.

Animals↗
PubMed1980

Relationship of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase Specific Activity to Subunit Composition.

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase, EC 4.1.1.39) was isolated from Nicotiana sylvestris and from two cultivars and three nuclear substitution lines of Nicotiana tabacum. Isoelectric focusing patterns, supported by amino acid analyses and tryptic peptide mapping, were used to divide these enzymes into two categories: (a) RuBPCase with variable large subunits and identical small subunits; and (b) RuBPCase with identical large but different small subunits. Specific activities for both the carboxylation and oxygenation reactions were determined for all six RuBPCase enzymes under standard conditions of activation and assay. High, intermediate, and low levels of carboxylase (880, 530, and 340 nanomoles HCO(3) (-) per milligram per minute) and oxygenase (66, 45, and 35 nanomoles O(2) per milligram per minute) activity were noted. The carboxylase to oxygenase ratios ranged from 9 to 14.

Journal Article↗
PubMed1990

Differential regulation of 5-lipoxygenase and cyclo-oxygenase pathways of arachidonate metabolism in rat peritoneal leukocytes.

1. Simultaneous activation of the 5-lipoxygenase and cyclo-oxygenase pathways of arachidonate metabolism in rat peritoneal mixed leukocytes in response to A23187, chemoattractant N-formyl-methionine-leucine-phenylalanine (FMLP) and arachidonic acid (AA) was studied by radioimmunoassay of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) respectively. 2. FMLP and AA preferentially activated cyclo-oxygenase and A23187 preferentially activated 5-lipoxygenase. Release of TXB2 preceded that of LTB4. A threshold amount of A23187 enhanced FMLP-and AA-induced LTB4 production but not that of TXB2. 3. Phorbol myristate acetate (PMA) abolished LTB4 generation in response to FMLP with much less effect on TXB2, but did not inhibit the formation of either eicosanoid caused by A23187 or AA. Instead, PMA caused a dose-dependent but modest stimulation of TXB2 generation either on its own or when added with A23187 or AA. 4. These results show that the 5-lipoxygenase and cyclo-oxygenase pathways in rat peritoneal leukocytes are regulated differently and that functional compartmentalisation of the stimulus-generation sequence operates in these cells.

Animals↗
PubMed1994

Production of PGE2 by bovine cultured airway smooth muscle cells and its inhibition by cyclo-oxygenase inhibitors.

1. Prostaglandin E2 (PGE2) is thought to be an important inhibitory modulator of inflammatory processes in the airway. It inhibits inflammatory cell function and cholinergic neurotransmission in vitro and roles have been postulated in vivo in refractoriness and in the mechanism of action of the diuretic agent, frusemide. 2. The production of PGE2 by bovine cultured airway smooth muscle cells has been studied under a range of conditions. The effects of cyclo-oxygenase inhibitors (flurbiprofen, indomethacin, acetyl salicylic acid) on serum-induced production of PGE2 were assessed over a range of concentrations (10(-7)-10(-4) M). 3. Serum-stimulated production of PGE2 in control wells ranged from 350 to 800 ng PGE2 ml-1 in cells from different animals. All three cyclo-oxygenase inhibitors inhibited PGE2 production with an order of potency, flurbiprofen > indomethacin > acetyl salicylic acid. Log IC50 values were -6.24 for flurbiprofen, -5.23 for indomethacin and -3.50 for acetyl salicylic acid. 4. PGE2 production was stimulated by arachidonic acid (10(-5) M) or addition of the proinflammatory mediator, bradykinin (10(-8)-10(-5) M). 5. Incubation of cells for 24 h with 5 bromo deoxyuridine (BRDU) (10(-4) M) to prevent DNA synthesis did not alter PGE2 production in response to serum, suggesting that it was not a function of proliferation per se. 6. Our study suggests that airway smooth muscle may be an important source of PGE2. Production of PGE2 may be a novel feedback mechanism whereby airway smooth muscle cells can negatively modulate airways inflammation. The differing potencies of the cyclo-oxygenase inhibitors may explain the contrasting effect of these drugs in recent studies in asthma.

Animals↗
PubMed1988

Selectivity of neutrophil 5-lipoxygenase and cyclo-oxygenase inhibition by an anti-inflammatory flavonoid glycoside and related aglycone flavonoids.

A newly described plant-derived flavonoid, hypolaetin-8-glucoside, which has anti-inflammatory and gastroprotective actions in-vivo, and its corresponding aglycone, hypolaetin, have been compared with 14 other flavonoids for inhibition of eicosanoid generation via the 5-lipoxygenase and cyclo-oxygenase pathways in elicited rat peritoneal leukocytes stimulated with calcium ionophore. Comparable results for the inhibitory profiles of the compounds were obtained using either radioimmunoassay of released eicosanoids or radio-TLC of metabolites formed from labelled arachidonate, but there were differences in absolute potency of the inhibitors. Hypolaetin-8-glucoside was a weak but selective inhibitor of 5-lipoxygenase (IC50 56 microM vs 5-lipoxygenase; greater than 1000 microM vs cyclo-oxygenase), whereas the aglycone hypolaetin was a more potent and selective 5-lipoxygenase inhibitor (IC50 4.5 microM vs 70 microM). Results with three other glycoside/aglycone pairs confirmed that addition of sugar residues greatly reduces inhibitory potency whilst retaining selectivity against 5-lipoxygenase. Analysis of 12 aglycone flavonoids showed that inhibitory potency and selectivity against 5-lipoxygenase is conferred by the presence of 3'4'-vicinal diol (catechol) in ring B as part of a 3,4-dihydroxycinnamoyl structure as proposed by others and by incorporation of additional hydroxyl substituents. In contrast, "cross-over" of inhibitory selectivity is observed in compounds containing few hydroxyl substituents (with none in ring B) which are selective against cyclo-oxygenase. These results are discussed in relation to possible mechanisms of hypolaetin-8-glucoside's protective actions and the concept that these inhibitory effects of flavonoids cannot be ascribed to a unitary free radical scavenging action.

Animals↗
PubMed1994

In vitro activation of cyclo-oxygenase in the rabbit carotid body: effect of its blockade on [3H]catecholamine release.

The release of prostaglandin E2 (PGE2) from rabbit carotid bodies (CBs) incubated in basal conditions (PO2 approximately 132 mmHg; PCO2 approximately 33 mmHg; pH = 7.42) amounts to 94.4 +/- 10.1 pg (mg protein)-1 (10 min)-1 (mean +/- S.E.M.). Incubation of the CB in a hypoxic solution (PO2 approximately 46 mmHg) produced a significant 40% increase (P < 0.05) in the release of PGE2. Indomethacin (2 microM) prevented the hypoxia-induced release of PGE2. Sensory plus sympathetic denervation of the CB 4 days prior to the experiments did not modify either basal or low PO2-induced PGE2 release, indicating that intraglomic nerve endings are not significant sources for the PGE2 released. Incubation of the CB in an acidic-hypercapnic solution (PO2 approximately 132 mmHg; PCO2 approximately 132 mmHg; pH = 6.60) or in a high K(+)-containing solution (35 mM) was also effective in promoting an increase in the outflow of PGE2 from the organs. The release of [3H]catecholamines ([3H]CA) from the CB elicited by incubating the organs in low PO2 solutions (PO2 ranged between 66 and 13 mmHg) was potentiated by two inhibitors of cyclo-oxygenase, acetylsalicylic acid (ASA, 100 microM) and indomethacin (2 microM). The effect persisted after chronic denervation of the organ. The secretory response elicited by acidic stimuli was also augmented by cyclo-oxygenase inhibitors. Thus, [3H]CA release elicited by incubating the CBs in the acidic-hypercapnic solution increased by 300% in the presence of indomethacin (2 microM), and ASA (100 microM) more than doubled the release induced by dinitrophenol (100 microM), a protonophore that mimics an acidic stimulus. Indomethacin, but not ASA, moderately increased the high K(+)-evoked [3H]CA release. The effect of indomethacin on the release of [3H]CA elicited by acidic and hypoxic stimuli was reversed by PGE2 in a dose-dependent manner (0.3-300 nM). These results show that low PO2 and high PCO2-low pH, the natural stimuli to the CB, as well as high extracellular [K+], activate the cyclo-oxygenase pathway in the CB, promoting an increase in the outflow of PGE2. The data also show that the blockade of this pathway activates the stimulus-induced [3H]CA release from the CB, indicating that naturally released prostanoids exert an inhibitory control on chemoreceptor cells. The data lend support to the notion that the hyper-reactivity of the ventilatory response to hypoxia in subjects under anti-inflammatory drug treatment results from CB cycloxygenase inhibition.

Animals↗
PubMed2006

Genetic characterization of the phenylacetyl-coenzyme A oxygenase from the aerobic phenylacetic acid degradation pathway of Escherichia coli.

We show here that the paaABCDE genes of the paa cluster responsible for phenylacetate degradation in Escherichia coli W encode a five-component oxygenase that hydroxylates phenylacetyl-coenzyme A (CoA), the first intermediate of the pathway. The primary structure of the subunits of bacterial phenylacetyl-CoA oxygenases revealed that these enzymes constitute the prototype of a new and distinct group of the large bacterial diiron multicomponent oxygenase family.

Acetyl Coenzyme A↗
PubMed1985

Complementation of a Rhodopseudomonas sphaeroides ribulose bisphosphate carboxylase-oxygenase regulatory mutant from a genomic library.

A genomic library containing HindIII partial digests of Rhodopseudomonas sphaeroides HR DNA was constructed in the broad-host-range cosmid cloning vector pVK102. With a portion of this library as donor in complementation studies with the ribulose 1,5-bisphosphate carboxylase-oxygenase regulatory mutant R. sphaeroides KW25/11, a fragment of DNA which is capable of partially complementing this mutant was isolated. In four independent matings, Aut+ transconjugants which contained a hybrid plasmid carrying the same 28-kilobase-pair insert were isolated. While complemented strains were capable of growing at rates equal to that of the wild type under photoautotrophic conditions, they were not able to match wild-type levels of ribulose 1,5-biphosphate carboxylase-oxygenase activity or of form I ribulose 1,5-bisphosphate carboxylase-oxygenase protein. In addition, there is some indication that recombination may be necessary for optimal complementation to occur. The size of the complementing fragment was further reduced to 2.7 kilobase pairs by using vectors constructed for subcloning.

Cloning, Molecular↗
PubMed1965

Synthesis of Protocatechuate Oxygenase by Pseudomonas fluorescens in the Presence of Exogenous Carbon Sources.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Synthesis of protocatechuate oxygenase by Pseudomonas fluorescens in the presence of exogenous carbon sources. J. Bacteriol. 90:15-22. 1965.-The addition of glucose, ribose, or fructose (0.45 or 45.0 mumoles/ml) simultaneously with protocatechuic acid shortens the lag period required for synthesis of protocatechuate oxygenase by a washed-cell suspension of Pseudomonas fluorescens. Glucose is readily oxidized and supports growth of P. fluorescens, whereas neither ribose nor fructose readily supports growth. High glucose concentrations (45.0 mumoles/ml) shorten the lag period but lower the total enzyme synthesis. The pH drops during glucose oxidation, and this is accompanied by a decrease in the rate of enzyme synthesis. High glucose concentrations, with adequate buffering, permitted "normal" enzyme synthesis. A decrease in the total enzyme synthesis was not observed in the presence of high concentrations of ribose or fructose. Succinate, pyruvate, acetate, or formate (0.45 mumole/ml) were readily oxidized, but did not shorten the lag period required for synthesis of the enzyme. The data suggest that glucose, ribose, or fructose may serve as a "specific" carbon source (such as ribose-5-phosphate or a similar precursor important in ribonucleic acid synthesis) functional in the synthesis of protocatechuate oxygenase.

Journal Article↗
PubMed2005

Risk of adverse gastrointestinal outcomes in patients taking cyclo-oxygenase-2 inhibitors or conventional non-steroidal anti-inflammatory drugs: population based nested case-control analysis.

OBJECTIVE: To determine the risk of an adverse upper gastrointestinal event in patients taking different cyclo-oxygenase-2 inhibitors compared with non-selective non-steroidal anti-inflammatory drugs. DESIGN: Nested case-control study. SETTING: 367 general practices contributing to the UK QRESEARCH database, spread throughout every strategic health authority and each health board in England, Wales, and Scotland. PARTICIPANTS: Patients aged 25 or more with a first ever diagnosis of an adverse upper gastrointestinal event (peptic ulcer or haematemesis) between 1 August 2000 and 31 July 2004 and up to 10 controls per case matched for age, sex, calendar time, and practice. MAIN OUTCOME MEASURES: Unadjusted and adjusted odds ratios for adverse upper gastrointestinal events associated with celecoxib, rofecoxib, ibuprofen, diclofenac, naproxen, other selective and non-selective non-steroidal anti-inflammatory drugs, and aspirin. RESULTS: The incidence of adverse upper gastrointestinal events was 1.36 per 1000 person years (95% confidence interval 1.34 to 1.39). We identified 9407 incident cases and 88 867 matched controls. Increased risks of adverse gastrointestinal events were associated with current use of cyclo-oxygenase-2 inhibitors and with conventional non-steroidal anti-inflammatory drugs. Risks were reduced after adjustment for confounders but remained significantly increased for naproxen (adjusted odds ratio 2.12, 95% confidence interval 1.73 to 2.58), diclofenac (1.96, 1.78 to 2.15), and rofecoxib (1.56, 1.30 to 1.87) but not for current use of celecoxib (1.11, 0.87 to 1.41). We found clinically important interactions with current use of ulcer healing drugs that removed the increased risks for adverse gastrointestinal events for all groups of non-steroidal anti-inflammatory drugs except diclofenac, which still had an increased odds ratio (1.49, 1.26 to 1.76). CONCLUSION: No consistent evidence was found of enhanced safety against gastrointestinal events with any of the new cyclo-oxygenase-2 inhibitors compared with non-selective non-steroidal anti-inflammatory drugs. The use of ulcer healing drugs reduced the increased risk of adverse gastrointestinal outcomes with all groups of non-steroidal anti-inflammatory drugs, but for diclofenac the increased risk remained significant.

Adult↗
PubMed1987

Synergistic enhancement of mitogen responses of human lymphocytes by inhibitors of cyclo-oxygenase and 5,8,11-eicosatriynoic acid, an inhibitor of 12-lipoxygenase and leukotriene biosynthesis.

Blood mononuclear cells are well-known producers of various cyclo-oxygenase and lipoxygenase metabolites of arachidonic acid, some of which possess immunoregulatory functions. In the present investigation, we have examined 3H-thymidine incorporation in human blood lymphocytes cultured with polyclonal mitogens and antigens in the presence of various inhibitors of cyclo-oxygenase (such as indomethacin and meclofenamic acid) and an inhibitor of 12-lipoxygenase and leukotriene biosynthesis, 5,8,11-eicosatriynoic acid (ETI). It was observed that these inhibitors could augment mitogen responses of nonpurified lymphocyte preparations but not of preparations which were depleted of monocytes. The results indicate that monocytes and not lymphocytes were the main producers of immunosuppressive eicosanoids derived from arachidonic acid. Further, mitogenic responses in the presence of both an inhibitor of cyclo-oxygenase and ETI were augmented to a higher extent than expected. Again, this enhancement was not observed in preparations depleted of monocytes. Arachidonic acid metabolism was examined in mitogen-stimulated cultures pulsed with 14C-arachidonic acid. It was observed that the production of three metabolites was inhibited by meclofenamic acid, whereas the amounts of another 14Cr-labeled compound were almost doubled in the presence of meclofenamic acid.

8,11,14-Eicosatrienoic Acid↗
PubMed2000

Effects of cyclo-oxygenase inhibition on vasodilatory response to acetylcholine in patients with type 1 diabetes and nondiabetic subjects.

OBJECTIVE: Studies examining vasodilatory responses to acetylcholine (ACh) and its derivatives have been conflicting. Enhanced activation of the cyclo-oxygenase pathway and increased availability of vasodilatory prostanoids may occur in type 1 diabetes, and this may compensate for the observed reduction in nitric oxide (NO) activity We examined the role of cyclo-oxygenase inhibition on vasodilatory responses in 12 healthy normotensive type 1 diabetic adults and 12 nondiabetic control subjects of similar age, sex, and BMI. RESEARCH DESIGN AND METHODS: Forearm blood flow was measured using a venous occlusion plethysmography technique at baseline and after brachial artery infusions of ACh (7.5, 15, and 30 microg/min). Forearm blood flow at baseline and after ACh was then reexamined after local intra-arterial infusion of indomethacin (0.3 mg/100 ml forearm volume), a cyclo-oxygenase inhibitor. RESULTS: Baseline blood flow in the diabetic and control groups were similar (2.65 +/- 0.26 vs. 2.59 +/- 0.20 ml/min per 100 ml, respectively; P = 0.4). After indomethacin infusion, the vasodilatory responses to all doses of ACh were reduced in both the diabetic (by 25.30 +/- 4.90%) and control group (by 11.23 +/- 5.45%). However, the reduction in blood flow response to ACh after indomethacin was greater in diabetic patients compared with control subjects (P = 0.03). CONCLUSIONS: Our findings suggest that vasodilatory, prostanoids are important in determining endothelial response to ACh in diabetic and nondiabetic subjects. Increased prostaglandin-mediated vasodilation may compensate for attenuated responses to NO previously reported in diabetic subjects. These findings may partly explain the conflicting reports of endothelial dysfunction in patients with type 1 diabetes.

Acetylcholine↗
PubMed1983

Anti-cyclo-oxygenase agents and asthma.

Aspirin inhibits cyclo-oxygenase by an irreversible time-dependent process of inactivation; this mechanism appears to be responsible for precipitation of asthmatic attacks in about 5%-10% of adult asthmatic patients. Besides aspirin, all other cyclo-oxygenase inhibitors induce bronchoconstriction in sensitive patients, while nonsteroidal anti-inflammatory drugs without anti-cyclo-oxygenase activity can be taken by the same patients with impunity. While aspirin-sensitive patients should avoid the potential for adverse reactions, there are rare reports of bronchodilation following ingestion of aspirin and aspirinlike drugs.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1992

Effect of lipoxygenase and cyclo-oxygenase inhibition on oleic acid-induced lung oedema.

The contribution of cyclo-oxygenase and 5-lipoxygenase metabolites on hemodynamics and oedema formation was investigated in 21 isolated rabbit lungs after a 10 min Oleic Acid (OA) infusion, by recording the changes on Fluid Filtration Rate (FFR) and Pulmonary Artery Pressure (PAP). Lungs (n = 7) were pre-treated with indomethacin (cyclo-oxygenase inhibitor) 50 min prior to OA or with Diethylcarbamazine (5-lipoxygenase inhibitor) (n = 7) or not pre-treated at all (control group, n = 7). The FFR in the indomethacin group was significantly greater than in the control and Diethylcarbamazine (DEC) groups 12 min after OA (7.6 +/- 2.3 mg.min-1 vs. 2.3 +/- 0.8 mg.min-1 and 0.96 +/- 0.8 mg.min-1 respectively) (P < 0.01). The FFR in the control lungs 20 min after OA was significantly greater than the corresponding DEC value (4.2 +/- 0.5 mg.min-1 vs. 1.6 +/- 1.0 mg.min-1) (P < 0.01). Mean Pulmonary Artery Pressure (MPAP) increased both in control and indomethacin groups (16.0 +/- 2.0 Torr to 24.3 +/- 3.7 Torr after 20 min OA and 14.4 +/- 2.5 Torr to 24.6 +/- 3.6 Torr at 10 min after OA, respectively), but MPAP value in DEC group did not significantly change 30 min after OA (14.7 +/- 1.5 Torr to 16.0 +/- 2.3 Torr) (P > 0.05). So we conclude that the selective inhibition of the 5-lipoxygenase metabolites (leukotriene-5hete) may play a protective role in OA induced oedema, whereas the selective inhibition of the cyclo-oxygenase pathway may have a deleterious effect on the hemodynamics and endothelial permeability in our experimental condition.

Animals↗
PubMed1988

Involvement of lipoxygenase and cyclo-oxygenase pathways in hypoxia and metabolic alkalosis produced by canatoxin in rats.

This paper reports on the metabolic alkalosis produced in rats by canatoxin, a neurotoxic principle extracted from Canavalia ensiformis seeds. Rats receiving canatoxin showed increased blood bicarbonate concentration with alkaline pH and no change in pCO2. A fall in pO2 was also seen. A dose-dependent relationship between pO2 decrease and bicarbonate increase was also observed with the toxin, suggesting that hypoxia may result from respiratory compensation. Canatoxin-induced hypoxia was both lipoxygenase and cyclo-oxygenase dependent but metabolic alkalosis was only blocked with cyclo-oxygenase inhibitors. Canatoxin activates the lipoxygenase pathway and probably increases leukotriene (LT) production. Since LT may release cyclo-oxygenase products, this may explain the metabolic alkalosis observed with canatoxin.

Acid-Base Equilibrium↗
PubMed1985

Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase.

The small subunits of spinach ribulosebisphosphate carboxylase-oxygenase were isolated by mild acid precipitation of the hexadecameric holoenzyme. About one-third of the small subunits remained in the supernatant while the remainder, and all of the large subunits, were precipitated and irreversibly denatured. The spinach small subunits were able to reassemble with the large subunit octamer of ribulosebisphosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus ACMM 323, prepared as described previously (Andrews, T. J., and Ballment, B. (1983) J. Biol. Chem. 258, 7514-7518) to produce a catalytically active, hybrid enzyme. The heterologous small subunits bound an order of magnitude less tightly than homologous small subunits and the specific activity of the hybrid, when fully saturated with foreign small subunits, was about half that of the homologously reassembled or native Synechococcus enzyme. In addition, the Km(CO2) of the hybrid was about twice as high. However, the degree of partitioning between carboxylation and oxygenation was identical for the hybrid, the homologously reassembled, and the native Synechococcus enzymes and clearly less in favor of carboxylation than partitioning by the spinach enzyme. Therefore, this important facet of catalysis by ribulosebisphosphate carboxylase-oxygenase appears to be specified exclusively by the large subunit.

Carbon Dioxide↗
PubMed1980

Interactions of hydrogen peroxide with ribulose bisphosphate carboxylase oxygenase.

Hydrogen peroxide inhibited both carboxylase and oxygenase activities of purified, and fully activated, spinach ribulose-1,5-bisphosphate (RuP2) carboxylase-oxygenase. Inhibition of the carboxylase reaction was mixed competitive with respect to CO2 (Ki = 1.2 mM) and uncompetitive with respect to RuP2. For the oxygenase reaction, H2O2 was a competitive inhibitor with respect to O2 (Ki = 2.1 mM) and an uncompetitive inhibitor with respect to RuP2. H2O2 did not alter the stoichiometry between CO2 and RuP2 in the carboxylase reaction, indicating that H2O2 was not itself a substrate for the enzyme. RuP2 decreased the rate of deactivation of the enzyme which occurred at limiting CO2 concentrations. H2O2 greatly enhanced this stabilizing effect of RuP2 but had no effect on the rate of deactivation in the absence of RuP2. The inhibitory and stabilizing effects of H2O2 varied similarly with H2O2 concentration. These instantaneous, reversible effects of H2O2 were readily distinguishable from an irreversible inhibitory effect which occurred quite slowly, and in the absence of RuP2. These observations are discussed in relation to the enzyme's catalytic mechanism and its activation-deactivation transformations.

Carbon Dioxide↗