The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the ‘essential’ Tyr9 by phenylalanine leads to a marked decrease in the kcat for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on kcat for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in kcat for CDNB, but an increase in kcat for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity (‘turnover number‘) for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

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Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

Biochemical Journal ◽

10.1042/bj3560217 ◽

2001 ◽

Vol 356 (1) ◽

pp. 217-222 ◽

Cited By ~ 5

Author(s):

Ricardo FRANCO ◽

Alice S. PEREIRA ◽

Pedro TAVARES ◽

Arianna MANGRAVITA ◽

Michael J. BARBER ◽

...

Keyword(s):

Absorption Spectra ◽

Catalytic Mechanism ◽

Protoporphyrin Ix ◽

Three Dimensional ◽

Iron Chelation ◽

Enzymic Activity ◽

Dimensional Structure ◽

Wild Type ◽

Wild Type Enzyme ◽

Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

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Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

Biochemical Journal ◽

10.1042/bj3590065 ◽

2001 ◽

Vol 359 (1) ◽

pp. 65-75 ◽

Cited By ~ 25

Author(s):

Valeria MENCHISE ◽

Catherine CORBIER ◽

Claude DIDIERJEAN ◽

Michele SAVIANO ◽

Ettore BENEDETTI ◽

...

Keyword(s):

Crystal Structure ◽

Proton Transfer ◽

Chlamydomonas Reinhardtii ◽

Catalytic Mechanism ◽

Structural Data ◽

Careful Analysis ◽

Wild Type ◽

Thioredoxin H ◽

Dynamics Simulations

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

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The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

Biochemical Journal ◽

10.1042/bj20021765 ◽

2003 ◽

Vol 372 (1) ◽

pp. 241-246 ◽

Cited By ~ 24

Author(s):

Chris NATHANIEL ◽

Louise A. WALLACE ◽

Jonathan BURKE ◽

Heini W. DIRR

Keyword(s):

Glutathione Transferase ◽

Conformational Stability ◽

Wild Type ◽

Catalytic Function ◽

Evolutionarily Conserved ◽

Glutathione S Transferases ◽

Equilibrium Unfolding ◽

The Stability ◽

Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

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

Biochemical Society Transactions ◽

10.1042/bst0290099 ◽

2001 ◽

Vol 29 (2) ◽

pp. 99-105 ◽

Cited By ~ 36

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).

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

Biochemical Journal ◽

10.1042/bj3270877 ◽

1997 ◽

Vol 327 (3) ◽

pp. 877-882 ◽

Cited By ~ 9

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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Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2008.02.006 ◽

2008 ◽

Vol 1784 (5) ◽

pp. 863-871 ◽

Cited By ~ 24

Author(s):

Rajesh K. Yadav ◽

Subhankar Dolai ◽

Swati Pal ◽

Subrata Adak

Keyword(s):

Cytochrome C ◽

Ascorbate Peroxidase ◽

Leishmania Major ◽

Kinetic Studies ◽

Wild Type ◽

Wild Type Enzyme

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Association of GSTO1 and GSTO2 Polymorphism with Risk of End-Stage Renal Disease Development and Patient Survival

Journal of Medical Biochemistry ◽

10.1515/jomb-2016-0009 ◽

2016 ◽

Vol 35 (3) ◽

pp. 302-311 ◽

Cited By ~ 5

Author(s):

Slavica Cimbaljevic ◽

Sonja Suvakov ◽

Marija Matic ◽

Marija Pljesa-Ercegovac ◽

Tatjana Pekmezovic ◽

...

Keyword(s):

Renal Disease ◽

End Stage Renal Disease ◽

Cardiovascular Complications ◽

Wild Type ◽

Reference Allele ◽

Kaplan Meier ◽

Glutathione S Transferases ◽

Stage Renal Disease ◽

End Stage

Summary Background: Oxidative stress in patients with end-stage renal disease (ESRD) is associated with long-term cardiovascular complications. The cytosolic family of glutathione S-transferases (GSTs) is involved in the detoxication of various toxic compounds and antioxidant protection. GST omega class members, GSTO1 and GSTO2 possess, unlike other GSTs, dehydroascorbate reductase and deglutathionylation activities. The aim of this study was to clarify the role of genetic polymorphisms of GSTO1 (rs4925) and GSTO2 (rs156697) as risk determinants for ESRD development, as well as in the survival of these patients. Methods: A total of 199 patients and 199 healthy subjects were included in the study and genotyped for both GSTO1 and GSTO2 polymorphism. Protein thiol and carbonyl groups as markers of protein oxidative damage were determined spectrophotometrically. Cox proportional hazard model and Kaplan-Meier analysis were performed to investigate the role of GSTO1 and GSTO2 genetic polymorphism on mortality of ESRD patients during the follow-up period (36 month). Results: Individuals carrying the variant GSTO2 GG genotype were at 2.45-fold higher risk of ESRD development compared to the wild type GSTO2 AA genotype (OR=2.45; 95%CI=1.18-5.07; p=0.016). The results of GSTO1/GSTO2 haplotype analysis showed that the haplotype combi - nation of GSTO1 (*A)/GSTO2 (*A) (GSTO1 variant/GSTO2 wild type allele) was protective for ESRD (OR=0.23 95%CI=0.12-0.44, p=0.001). Patients carrying at least one GSTO1 reference allele have shorter mean overall (Log rank=2.844, p =0.241) and cardiovascular survival probability (Log rank=4.211, p=0.122). Conclusions: GSTO polymorphisms have been shown to act as significant markers in assessing the risk of ESRD development and patients’ survival.

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Critical Role of Interdomain Interactions in the Conformational Change and Catalytic Mechanism of Endoplasmic Reticulum Aminopeptidase 1

Biochemistry ◽

10.1021/acs.biochem.6b01170 ◽

2017 ◽

Vol 56 (10) ◽

pp. 1546-1558 ◽

Cited By ~ 19

Author(s):

Athanasios Stamogiannos ◽

Zachary Maben ◽

Athanasios Papakyriakou ◽

Anastasia Mpakali ◽

Paraskevi Kokkala ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Conformational Change ◽

Catalytic Mechanism ◽

Critical Role ◽

Endoplasmic Reticulum Aminopeptidase 1 ◽

Interdomain Interactions

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Role of βAsn-243 in the Phosphate-binding Subdomain of Catalytic Sites ofEscherichia coliF1-ATPase

Journal of Biological Chemistry ◽

10.1074/jbc.m407608200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 46057-46064 ◽

Cited By ~ 28

Author(s):

Zulfiqar Ahmad ◽

Alan E. Senior

Keyword(s):

Atpase Activity ◽

Atp Synthase ◽

Catalytic Mechanism ◽

Wild Type ◽

Phosphate Binding ◽

Γ Subunit ◽

Catalytic Sites ◽

The Face ◽

Extensive Involvement

In the catalytic mechanism of ATP synthase, phosphate (Pi) binding and release steps are believed to be correlated to γ-subunit rotation, and Pibinding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue βAsn-243 appears centrally located in the Pi-binding subdomain of catalytic sites. Here we studied the role of βAsn-243 inEscherichia coliATP synthase by mutagenesis to Ala and Asp. Mutation βN243A caused 30-fold impairment of F1-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and Piprotected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. βN243D F1-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated βN243D F1-ATPase, and Pidid not affect activation. We conclude that residue βAsn-243 is not involved in Pibinding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the “attacking water” and of an associated second water.

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