The role of distal tryptophan in the bifunctional activity of catalase-peroxidases

2001 ◽  
Vol 29 (2) ◽  
pp. 99-105 ◽  
Author(s):  
G. Regelsberger ◽  
C. Jakopitsch ◽  
P. G. Furtmüller ◽  
F. Rueker ◽  
J. Switala ◽  
...  
Keyword(s):  
Catalase Activity ◽  
Intermediate Compound ◽  
Stopped Flow ◽  
Wild Type ◽  
Compound I ◽  
Wild Type Enzyme ◽  
Catalase Peroxidase ◽  
Electron Reduction ◽  
The One

Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).

scholarly journals Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

2004 ◽  
Vol 279 (44) ◽  
pp. 46082-46095 ◽  
Author(s):  
Christa Jakopitsch ◽  
Anabella Ivancich ◽  
Florian Schmuckenschlager ◽  
Anuruddhika Wanasinghe ◽  
Gerald Pöltl ◽  
...  
Keyword(s):  
Catalase Activity ◽  
Transient State ◽  
Wild Type ◽  
Deuterium Labeling ◽  
Wild Type Enzyme ◽  
Radical Species ◽  
Distal Side ◽  
In The Wild ◽  
Catalase Peroxidase ◽  
Heme Peroxidases

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp122, Tyr249, and Met275(SynechocysisKatG numbering). The known crystal structures suggest that Tyr249and Met275could be within hydrogen-bonding distance to Arg439. To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg439variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr249and Trp122. In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por·+→ Trp· (or Trp·+) → Tyr·. The M275I variant did not form the Trp· species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp122–Tyr249adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.

scholarly journals Redox status of cysteines does not alter functional properties of human dUTPase but the Y54C mutation involved in monogenic diabetes decreases protein stability

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Judit Eszter Szabó ◽  
Kinga Nyíri ◽  
Dániel Andrási ◽  
Judit Matejka ◽  
Olivér Ozahonics ◽  
...  
Keyword(s):  
Bone Marrow Failure ◽  
Thiol Group ◽  
Redox Status ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Redox Switch ◽  
The One ◽  
Thermal Stability Of ◽  
Potential Regulatory Role

AbstractRecently it was proposed that the redox status of cysteines acts as a redox switch to regulate both the oligomeric status and the activity of human dUTPase. In a separate report, a human dUTPase point mutation, resulting in a tyrosine to cysteine substitution (Y54C) was identified as the monogenic cause of a rare syndrome associated with diabetes and bone marrow failure. These issues prompt a critical investigation about the potential regulatory role of cysteines in the enzyme. Here we show on the one hand that independently of the redox status of wild-type cysteines, human dUTPase retains its characteristic trimeric assembly and its catalytic activity. On the other hand, the Y54C mutation did not compromise the substrate binding and the catalytic properties of the enzyme at room temperature. The thermal stability of the mutant protein was found to be decreased, which resulted in the loss of 67% of its activity after 90 min incubation at the physiological temperature in contrast to the wild-type enzyme. In addition, the presence or absence of reducing agents had no effect on hDUTY54C activity and stability, although it was confirmed that the introduced cysteine contains a solvent accessible thiol group.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

Effect of Small, Acid-Soluble Proteins on Spore Resistance and Germination under a Combination of Pressure and Heat Treatment

2007 ◽  
Vol 70 (9) ◽  
pp. 2168-2171
Author(s):  
JONG-KYUNG LEE ◽  
SARA MOVAHEDI ◽  
STEPHEN E. HARDING ◽  
BERNARD M. MACKEY ◽  
WILLIAM M. WAITES
Keyword(s):  
Heat Treatment ◽  
High Pressure ◽  
High Temperature ◽  
Spore Germination ◽  
Soluble Proteins ◽  
Wild Type ◽  
Spore Resistance ◽  
Pressure Resistance ◽  
The One

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of α/β-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65°C and 100 to 500 MPa) and applied to wild-type and SASP-α−/β− Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65°C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65°C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65°C inactivated wild-type SASP-α+/β+ spores more than mutant SASP-α−/β− spores, and this was attributed to less pressure-induced germination in SASP-α−/β− spores than in wild-type SASP-α+/β+ spores. However, there was no difference in the pressure resistance between SASP-α+/β+ and SASP-α−/β− spores at 100 MPa and ambient temperature (approximately 22°C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that α/β-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.

Catalase activity in Campylobacter jejuni: comparison of a wild-type strain with an aerotolerant variant

1990 ◽  
Vol 36 (6) ◽  
pp. 449-451 ◽  
Author(s):  
Pamela A. Vercellone ◽  
Robert M. Smibert ◽  
Noel R. Krieg
Keyword(s):  
Campylobacter Jejuni ◽  
Catalase Activity ◽  
Specific Activity ◽  
Wild Type ◽  
Oxygen Tolerance ◽  
Variant Strain ◽  
Cultural Conditions ◽  
Cell Extracts ◽  
Bactericidal Effects

A comparison of Campylobacter jejuni VPI strain H840 (ATCC 29428), which can grow at O2 levels up to 15%, with variant strain MC711-01 (which can grown at O2 levels up to 21–26%) indicated that the specific activity of catalase in crude cell extracts was higher in the variant by a factor of 1.6 to 2.5, depending on cultural conditions. Smaller differences occurred with superoxide dismutase activity, while peroxidase activities were invariably lower in the variant strain. The variant strain was much more resistant than the wild type to the bactericidal effects of H2O2. The results suggest that catalase activity might be one of the factors associated with the greater tolerance of O2 by the variant strain. However, both strains became more susceptible to H2O2 when cultures were initially grown at 6% O2 and then shifted to 21% O2; thus the role of catalase in the oxygen tolerance of C. jejuni is probably minor. Key words: Campylobacter jejuni, catalase, oxygen tolerance.

ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
G A Vehar ◽  
K M Tate ◽  
D L Higgins ◽  
W E Holmes ◽  
H L Heyneker
Keyword(s):  
Cho Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Single Chain ◽  
Serine Protease Family ◽  
The One ◽  
Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

scholarly journals Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

1992 ◽  
Vol 285 (1) ◽  
pp. 187-192 ◽  
Author(s):  
C S Miles ◽  
N Rouvière-Fourmy ◽  
F Lederer ◽  
F S Mathews ◽  
G A Reid ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Isotope Effects ◽  
Mutant Enzyme ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flavocytochrome B2 ◽  
Rate Determining Step ◽  
Kinetic Isotope

The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

Hyperoxia increases H2O2 production by brain in vivo

1987 ◽  
Vol 63 (1) ◽  
pp. 353-358 ◽  
Author(s):  
T. Yusa ◽  
J. S. Beckman ◽  
J. D. Crapo ◽  
B. A. Freeman
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Catalase Activity ◽  
Intermediate Compound ◽  
H2o2 Production ◽  
H2o2 Concentration ◽  
Compound I ◽  
Central Nervous System Toxicity ◽  
Competitive Substrate

Hyperoxia and hyperbaric hyperoxia increased the rate of cerebral hydrogen peroxide (H2O2) production in unanesthetized rats in vivo, as measured by the H2O2-mediated inactivation of endogenous catalase activity following injection of 3-amino-1,2,4-triazole. Brain catalase activity in rats breathing air (0.2 ATA O2) decreased to 75, 61, and 40% of controls due to endogenous H2O2 production at 30, 60, and 120 min, respectively, after intraperitoneal injection of 3-amino-1,2,4-triazole. The rate of catalase inactivation increased linearly in rats exposed to 0.6 ATA O2 (3 ATA air), 1.0 ATA O2 (normobaric 100% O2) and 3.0 ATA O2 (3 ATA 100% O2) compared with 0.2 ATA O2 (room air). Catalase inactivation was prevented by pretreatment of rats with ethanol (4 g/kg), a competitive substrate for the reactive catalase-H2O2 intermediate, compound I. This confirmed that catalase inactivation by 3-amino-1,2,4-triazole was due to formation of the catalase-H2O2 intermediate, compound I. The linear rate of catalase inactivation allows estimates of the average steady-state H2O2 concentration within brain peroxisomes to be calculated from the formula: [H2O2] = 6.6 pM + 5.6 ATA-1 X pM X [O2], where [O2] is the concentration of oxygen in ATA that the rats are breathing. Thus the H2O2 concentration in brains of rats exposed to room air is calculated to be about 7.7 pM, rises 60% when O2 tension is increased to 100% O2, and increases 300% at 3 ATA 100% O2, where symptoms of central nervous system toxicity first become apparent. These studies support the concept that H2O2 is an important mediator of O2-induced injury to the central nervous system.

scholarly journals Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group

1997 ◽  
Vol 327 (3) ◽  
pp. 877-882 ◽  
Author(s):  
Junutula Reddy JAGATH ◽  
Naropantul APPAJI RAO ◽  
Handanahal SubbaRao SAVITHRI
Keyword(s):  
Carboxy Group ◽  
Gel Filtration ◽  
Serine Hydroxymethyltransferase ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Partial Dissociation ◽  
Tetrameric Form

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5ʹ-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, β-phenylserine or D-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4×10-4 s-1 at 50 μM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 μM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 μM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 μM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 °C) than that of the wild-type enzyme (56 °C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently ‘open’ form and the increased apparent Tm could be due to enhanced subunit interactions.

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