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Characterization of the Amino Acids from Neisseria meningitidis MsrA Involved in the Chemical Catalysis of the Methionine Sulfoxide Reduction Step

2006; Elsevier BV; Volume: 281; Issue: 51 Linguagem: Inglês

10.1074/jbc.m608844200

1083-351X

Mathias Antoine, Adeline Gand, Sandrine Boschi‐Müller, Guy Branlant,

Enzyme Structure and Function

Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that reduce protein-bound methionine sulfoxide back to Met in the presence of thioredoxin. In vivo, the role of the Msrs is described as essential in protecting cells against oxidative damages and as playing a role in infection of cells by pathogenic bacteria. There exist two structurally unrelated classes of Msrs, called MsrA and MsrB, specific for the S and the R epimer of the sulfoxide function of methionine sulfoxide, respectively. Both Msrs present a similar catalytic mechanism, which implies, as a first step, a reductase step that leads to the formation of a sulfenic acid on the catalytic cysteine and a concomitant release of a mole of Met. The reductase step has been previously shown to be efficient and not rate-limiting. In the present study, the amino acids involved in the catalysis of the reductase step of the Neisseria meningitidis MsrA have been characterized. The invariant Glu-94 and to a lesser extent Tyr-82 and Tyr-134 are shown to play a major role in the stabilization of the sulfurane transition state and indirectly in the decrease of the pKapp of the catalytic Cys-51. A scenario of the reductase step is proposed in which the substrate binds to the active site with its sulfoxide function largely polarized via interactions with Glu-94, Tyr-82, and Tyr-134 and participates via the positive or partially positive charge borne by the sulfur of the sulfoxide in the stabilization of the catalytic Cys. Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that reduce protein-bound methionine sulfoxide back to Met in the presence of thioredoxin. In vivo, the role of the Msrs is described as essential in protecting cells against oxidative damages and as playing a role in infection of cells by pathogenic bacteria. There exist two structurally unrelated classes of Msrs, called MsrA and MsrB, specific for the S and the R epimer of the sulfoxide function of methionine sulfoxide, respectively. Both Msrs present a similar catalytic mechanism, which implies, as a first step, a reductase step that leads to the formation of a sulfenic acid on the catalytic cysteine and a concomitant release of a mole of Met. The reductase step has been previously shown to be efficient and not rate-limiting. In the present study, the amino acids involved in the catalysis of the reductase step of the Neisseria meningitidis MsrA have been characterized. The invariant Glu-94 and to a lesser extent Tyr-82 and Tyr-134 are shown to play a major role in the stabilization of the sulfurane transition state and indirectly in the decrease of the pKapp of the catalytic Cys-51. A scenario of the reductase step is proposed in which the substrate binds to the active site with its sulfoxide function largely polarized via interactions with Glu-94, Tyr-82, and Tyr-134 and participates via the positive or partially positive charge borne by the sulfur of the sulfoxide in the stabilization of the catalytic Cys. Methionine sulfoxide reductases (Msr) 3The abbreviations used are: Msr, methionine sulfoxide reductase; 2PDS, 2,2′-dipyridyl disulfide; AcMetSONHMe, Ac-l-Met-R,S-SO-NHMe; AcMet-NHMe, Ac-l-Met-NHMe; Me2SO, dimethyl sulfoxide; MetSO, methionine sulfoxide; pKapp, apparent pKa; Trx, thioredoxin. 3The abbreviations used are: Msr, methionine sulfoxide reductase; 2PDS, 2,2′-dipyridyl disulfide; AcMetSONHMe, Ac-l-Met-R,S-SO-NHMe; AcMet-NHMe, Ac-l-Met-NHMe; Me2SO, dimethyl sulfoxide; MetSO, methionine sulfoxide; pKapp, apparent pKa; Trx, thioredoxin. are enzymes that catalyze the reduction of free and protein-bound methionine sulfoxide (MetSO) back to Met. Two structurally unrelated classes of Msrs have been described so far. MsrAs are stereo specific toward the S isomer on the sulfur of the sulfoxide function, whereas MsrBs are specific toward the R isomer. Both classes share a similar three-step catalytic mechanism (Scheme 1). First, the reductase step leads to formation of a sulfenic acid intermediate on the catalytic cysteine concomitantly with the release of one mole of Met/mole of Msr. Then, an intra-disulfide bond is formed via the attack of a second Cys (called the recycling Cys) on the sulfenic acid intermediate accompanied by release of a water molecule. Finally, the disulfide bond is reduced by thioredoxin (Trx) in the last step. Recently, the kinetics of the three steps have been investigated for MsrA and MsrB domains of the PilB protein of Neisseria meningitidis (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 2Olry A. Boschi-Muller S. Branlant G. Biochemistry. 2004; 43: 11616-11622Crossref PubMed Scopus (54) Google Scholar). For both classes of Msrs, the rate-limiting step is associated with the Trx recycling process, whereas the rate of formation of the intra-disulfide bond is governed by that of formation of the sulfenic acid intermediate, the rate of which is fast.The three-dimensional structures of the MsrA from Escherichia coli, Bos taurus, and Mycobacterium tuberculosis have been recently solved by x-ray crystallography (3Taylor A.B. Benglis Jr., D.M. Dhandayuthapani S. Hart P.J. J. Bacteriol. 2003; 185: 4119-4126Crossref PubMed Scopus (67) Google Scholar, 4Tete-Favier F. Cobessi D. Boschi-Muller S. Azza S. Branlant G. Aubry A. Structure Fold Des. 2000; 8: 1167-1178Abstract Full Text Full Text PDF Scopus (83) Google Scholar, 5Lowther W.T. Brot N. Weissbach H. Matthews B.W. Biochemistry. 2000; 39: 13307-13312Crossref PubMed Scopus (120) Google Scholar). The active site can be represented as an opened basin readily accessible to the MetSO substrate in which the catalytic Cys-51 is located at the entrance of the α 1 helix. In all the structures, the active site is occupied by a molecule that is covalently or non-covalently bound to the catalytic cysteine. In the case of E. coli MsrA, a dimethyl arsenate molecule is covalently bound, whereas it is a dithiothreitol molecule in B. taurus enzyme. In the case of M. tuberculosis MsrA, a methionine residue from a neighboring monomer occupies the active site. In all three structures, a water molecule is present, the position of which can mimic the oxygen atom of the sulfoxide function of MetSO. This water molecule is tightly H-bonded to three invariant amino acid residues, i.e. Tyr-82, Glu-94, and Tyr-134. All the three structures also support the involvement of invariant Phe-52 and Trp-53 in the substrate recognition via the formation of a hydrophobic pocket in which the ϵ methyl group of MetSO can bind.Study of the reduction mechanism of dimethyl sulfoxide (Me2SO) by methanethiol in Me2SO solution has recently been investigated by quantum chemistry calculations (6Balta B. Monard G. Ruiz-Lopez M.F. Antoine M. Gand A. Boschi-Muller S. Branlant G. J. Phys. Chem. A. 2006; 110: 7628-7636Crossref PubMed Scopus (31) Google Scholar). It was shown that 1) a sulfurane species is formed prior to formation of either a sulfenic acid intermediate or a disulfide species and 2) the rate-limiting step is governed by proton transfer between the thiol and the sulfoxide functions prior to sulfurane formation. Although these conclusions are derived from studies based on a model in solution, they provide a framework for the study of the chemical reductase step occurring within the MsrA active site.In the present study, the role of Glu-94, Tyr-82, and Tyr-134 residues and how the catalytic Cys-51 is stabilized in the reductase step of the MsrA from N. meningitidis have been investigated. For that, the kinetic parameters and the pH dependence of the rate constant of the reductase step of mutated MsrAs at positions 82, 94, and 134 were determined and compared with those of the wild type. The pKapp of Cys-51 in the free enzyme was also determined. The results show that Cys-51 is activated upon substrate binding to the active site with a shift of its pKa from 9.5 to 5.7. Substitutions at positions 82, 94, and 134 do not modify the apparent affinity for the substrate in the reductase step. In contrast, drastic decrease of the reductase step rate is observed for the E94A and Y82F/Y134F MsrAs, whereas E94Q MsrA displays only a small decrease. Moreover, each mutated MsrA is characterized by a shift of the pKapp of its Cys-51 to higher values compared with wild type. Taking into account all the results, a scenario for the catalysis of the sulfoxide reductase step is proposed in which Glu-94, Tyr-82, and Tyr-134 stabilize the sulfurane transition state formed. In this scenario, the substrate binds to the active site with its sulfoxide function largely polarized via interactions with the side chains of Glu-94, Tyr-82, and Tyr-134 and plays a major role in stabilizing Cys-51 via the positive, or partially positive, charge borne by the sulfur of the sulfoxide function.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis, Production, and Purification of Wild-type and Mutated N. meningitidis MsrAs—The E. coli strain used for all N. meningitidis MsrA productions was BE002 (MG1655 msrA::specΩ, msrB::α 3kana), transformed with the plasmidic construction pSKPILBMsrA containing only the coding sequence of msrA from pilB, under the lac promoter (7Olry A. Boschi-Muller S. Marraud M. Sanglier-Cianferani S. Van Dorsselear A. Branlant G. J. Biol. Chem. 2002; 277: 12016-12022Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The BE002 strain was kindly provided by Dr. F. Barras. Its use prevented expression of endogenous wild-type MsrA and MsrB from E. coli and thus avoided any contamination of the activity of the N. meningitidis MsrA by the Msrs from E. coli. Site-directed mutageneses were performed using the QuikChange site-directed mutagenesis kit (Stratagene).Purifications were realized as previously described (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Wild-type and mutated MsrAs were pure, as checked by electrophoresis on 12.5% SDS-PAGE gel followed by Coomassie Brilliant Blue R-250 staining and by electrospray mass spectrometry analyses. Storage of the enzymes was done as previously described. The molecular concentration was determined spectrophotometrically, using extinction coefficient at 280 nm of 26,200 m–1·cm–1 for wild-type and mutated MsrAs. In this report, N. meningitidis MsrA amino acid numbering is based on E. coli MsrA sequence.Quantification of the Free Cysteine Content with 5,5′-Dithiobis(2-nitro)benzoate—Cysteine content of MsrA was routinely determined using 5,5′-dithiobis(2-nitro)benzoate under non-denaturing conditions in buffer A (50 mm Tris-HCl, 2 mm EDTA, pH 8) as previously described (8Boschi-Muller S. Azza S. Sanglier-Cianferani S. Talfournier F. Van Dorsselear A. Branlant G. J. Biol. Chem. 2000; 275: 35908-35913Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar).pH Dependence of MsrA Thiol Reaction Rates with 2,2′-Dipyridyl Disulfide (2PDS)—Because of the high reactivity of Cys-51 and Cys-198 in MsrA, fast kinetic measurements were carried out on an Applied PhotoPhysics SX18MV-R stopped-flow apparatus. MsrA reactions with 2PDS were performed at 25 °C under pseudo-first-order conditions in 30 mm acetic acid, 30 mm imidazole, 120 mm Tris/HCl buffer at constant ionic strength of 0.15 m over a pH range of 6 to 10 (polybuffer B). MsrA and 2PDS concentrations after mixing were 6.2 and 310 μm, respectively. The pseudo-first-order rate constant kobs was determined at each pH by fitting the absorbance (A) at 343 nm versus time (t) to mono-exponential Equation 1, where a is the burst amplitude and c is the end point. The second-order rate constants k2 were calculated by dividing kobs by 2PDS concentration and then fitted to Equation 2, in which k2max represents the second rate constant for the thiolate form. A=a(1-e-kobs t)+c(Eq. 1) k2=k2max1+10(pKa-pH)(Eq. 2) Measurement of the Thiol Ionization by Ultraviolet Absorbance—Absorbance spectra were measured for all enzymes in 1.0-cm path length quartz cuvettes in a SAFAS UV-visible absorbance spectrophotometer. The protein samples were diluted to 23 μm in polybuffer B. Spectra were recorded at 25 °C in 0.5-nm steps from 300 to 200 nm over a pH range of 7 to 10. The buffer solution was scanned relative to air, followed by a protein solution in the same cuvette versus air. The two spectra were then subtracted and the difference converted to molar absorption coefficients at 240 nm (ϵ240 nm). Data were fitted to a model derived from the Henderson-Hasselbach equation as shown in Equation 3 for one apparent pKa. ϵ240nm=ϵSH+ϵS-1+10(pKa-pH)(Eq. 3) Steady-state MsrA Kinetics in the Presence of the Trx Recycling System—Steady-state kinetic parameters were determined with the Trx reductase recycling system (E. coli Trx (100 μm), E. coli Trx reductase (4.8 μm), NADPH (1.2 mm)) and by varying the concentrations of AcMetSONHMe. AcMetSONHMe was prepared and purified as previously described (2Olry A. Boschi-Muller S. Branlant G. Biochemistry. 2004; 43: 11616-11622Crossref PubMed Scopus (54) Google Scholar). Initial rate measurements were carried out at 25 °C in buffer A or polybuffer B on a Kontron Uvikon 933 spectrophotometer by following the decrease of the absorbance at 340 nm due to the oxidation of NADPH. Initial rate data were fitted to the Michaelis-Menten relationship using least squares analysis to determine kcat and Km for AcMetSONHMe. E. coli Trx1 and Trx reductase were prepared following experimental procedures already published (7Olry A. Boschi-Muller S. Marraud M. Sanglier-Cianferani S. Van Dorsselear A. Branlant G. J. Biol. Chem. 2002; 277: 12016-12022Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).Preparation of MsrA under Oxidized Disulfide State—MsrA oxidation was achieved by mixing 100 μm MsrA with 100 mm MetSO in buffer A. The MetSO used was DL-Met-R,S-SO of which only the S isomer is a substrate for MsrA. After 10 min of incubation at room temperature, oxidized proteins were passed through an Econo-Pac 10 DG desalting column (Bio-Rad) equilibrated with buffer A. Oxidation of MsrA in the disulfide state was checked by titration with 5,5′-dithiobis(2-nitro)benzoate.Fluorescence Properties of Wild-type and Mutated MsrAs—The fluorescence excitation and emission spectra of wild-type and mutated MsrAs in their reduced and Cys-51/Cys-198 disulfide state were recorded on a flx spectrofluorometer (SAFAS) thermostated at 25 °C in buffer A with 10 μm of each protein as previously described (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).Determination of the Rate of Met Formation and of Thiol Loss by Single Turnover Quenched Flow Experiments—Quenched flow measurements were carried out at 25 °C on a SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) fitted for double mixing and adapted to recover the quenched samples as previously described (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The apparatus worked in a pulsed mode. Under the conditions used, a minimum aging time of ∼25–40 ms was determined. Equal volumes (57.5 μl) of a solution containing 550 μm Glu-94-mutated MsrA in buffer A and a solution containing AcMetSONHMe in buffer A were mixed in the aging loop. The mixture was then allowed to react for the desired time before being mixed with 115 μl of a quenched aqueous solution containing 2% of trifluoroacetic acid. Quenched samples were then collected in a 200-μl loop. For each aging time, four shots were done and the four corresponding quenched samples were pooled in a volume of 700 μl and then analyzed.After protein precipitation and centrifugation, Ac-l-MetNHMe (AcMetNHMe) quantification in the resulting supernatant was carried out by reverse phase chromatography as previously described (2Olry A. Boschi-Muller S. Branlant G. Biochemistry. 2004; 43: 11616-11622Crossref PubMed Scopus (54) Google Scholar): 100 μl were injected onto a 4.6 × 250-mm Atlantis dC18 reverse phase column (Waters) on an AKTA explorer system (Amersham Biosciences) equilibrated with H2O/0.1% trifluoroacetic acid. AcMetNHMe was eluted after AcMetSONHMe with a linear gradient of acetonitrile.The other part of the quenched samples that was not treated with 100% of trifluoroacetic acid was used to 1) determine the protein concentration from the absorbance at 280 nm and 2) quantify the free cysteine content, using 2PDS as a thiol probe, in the presence of urea to avoid precipitation of the protein in the cuvette. Progress curves of pyridine-2-thione production were recorded at 343 nm in 1.1 m urea, buffer A. Enzyme concentration was 6.19 μm, and 2PDS concentration was 665 μm. The amount of pyridine-2-thione formed was calculated using an extinction coefficient at 343 nm of 8,080 m–1·cm–1.Data were plotted as mole of AcMetNHMe formed/mole of MsrA and as free remaining thiols/mole of MsrA, both as a function of time. The rate of Met formation was determined by fitting the curve to the monoexponential Equation 4 in which a represents the fraction of Met formed/mole of MsrA and kMet represents the rate constant. y=a(1-e-kMett)(Eq. 4) The rate of loss in free thiols was determined by fitting the curve to the monoexponential Equation 5 in which y0 represents the number of free remaining thiols, a the number of oxidized thiols, and kSS the rate constant. y=y0+ae-kSSt(Eq. 5) Kinetics of the Formation of the Cys-51/Cys-198 MsrA Disulfide Bond in the Absence of Reductant by Single Turnover Stopped-flow Experiment at pH 8—Kinetics of the Trp-53 fluorescence variation associated with the formation of the Cys-51/Cys-198 disulfide bond were measured for E94Q, Y82F, and Y134F MsrAs at 25 °C on a SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) fitted for fluorescence measurements as described previously (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The excitation wavelength was set at 284 nm, and the emitted light was collected using a 320-nm cutoff filter. One syringe contained MsrA in buffer A (10 μm final concentration after mixing), and the other one contained AcMetSONHMe at various concentrations in buffer A. An average of at least six runs was recorded for each AcMetSONHMe concentration. Rate constants, kobs, were obtained by fitting fluorescence traces with the monoexponential Equation 6 in which c represents the end point, a the amplitude of the fluorescence increase (<0), and kobs the rate constant. y=ae-kobst+c(Eq. 6) Data were fitted to Equation 7 using least square analysis to determine kmax and KS for AcMetSONHMe. S represents the AcMetSONHMe concentration and KS the apparent affinity constant. kobs=kmaxSKS+S(Eq. 7) Kinetics of the Trp-53 fluorescence variation associated with the formation of the Cys-51/Cys-198 disulfide bond were measured for Y82F/Y134F and Y82F/Y134F/E94Q MsrAs at 25 °C on a flx spectrofluorometer (SAFAS). The excitation wavelength was set at 284 nm, and the fluorescence emission at 340 nm was recorded versus time after enzyme addition. Data were then treated as described above to obtain kobs, kmax, and KS values.pH Dependence of the Reductase Step Rate Constant—Determination of kmax and KS as a function of pH was carried out for wild-type MsrA by single turnover pre-steady-state fluorescence stopped-flow spectroscopy, using the same procedure as described in the previous section but replacing buffer A with polybuffer B. kobs values for E94Q, Y82F, Y134F, Y82F/Y134F, and Y82F/Y134F/E94Q MsrAs were determined at saturating concentration of AcMetSONHMe as a function of pH. Kinetics of Trp-53 fluorescence variation were recorded either with the stopped-flow apparatus or the spectrofluorometer depending on the mutated MsrA, as described in the previous section. The pH dependence of the reductase step rate constant for E94A and E94D MsrAs was determined under steady-state conditions using the Trx recycling system. kmax (or kobs) values were plotted against pH and fitted to Equation 8, deriving from a one-pKa model, where kmax opt represents the maximum pH-independent rate constant. kmax=kmaxopt(1+10(pKa-pH))(Eq. 8) RESULTSDetermination of pKapp of the Cys ResiduesThe pKapp of both Cys-51 and Cys-198 were determined in the reduced free enzyme by two methods. The first one involved determining the second-order rate constant of the reaction with the Cys-specific reactivity probe 2-PDS as a function of pH. The second one took advantage of the variation of the thiolate UV absorbance as a function of pH.Kinetics of Reaction of Reduced Wild-type, C51S, and C198S MsrAs with 2PDS—Reaction of 2PDS with wild-type MsrA obeyed pseudo-first-order kinetics, with formation of 2 mol of pyridine-2-thione/mol of MsrA as determined from the absorbance change at 343 nm. This result was expected as two Cys are present in N. meningitidis MsrA at positions 51 and 198. For all pH used, stopped-flow traces fitted to monoexponential Equation 1, with amplitude corresponding to the release of 2 mol of pyridine-2-thione. pH-k2 profile fitted to monosigmoidal Equation 2 with a pKapp value of 9.7 and k2max value of (2.4 ± 0.3)·105 m–1 s–1 (Fig. 1A). The product of 2PDS reaction with wild-type MsrA is the disulfide-oxidized enzyme and not the thiopyridine adducts. Indeed, no release of pyridine-2-thione was observed when 10 mm dithiothreitol was added to the purified product (data not shown).FIGURE 1pH dependence of the second-order rate constant k2 for the reaction of the thiol group with 2PDS of wild-type (•, panel A), C51S (□, panel B) and C198S (Δ, panel B) MsrAs. Reaction kinetics were performed at 25 °C over a pH range of 5–9 or 10 in polybuffer B. The concentrations of enzymes and 2PDS were 6.2 and 310 μm, respectively. Values of kobs were determined using nonlinear regression analysis, and second-order rate constants k2 were fitted to Equation 2 (solid line) (see also "Experimental Procedures"). The plateau of the sigmoidal plot was not attained at the pH tested. Therefore, the pKapp values obtained by fitting could only be taken as estimates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)C51S and C198S MsrAs behaved similarly to wild-type MsrA, except that only 1 mol of pyridine-2-thione/mol of MsrA was formed. pKapp value of 9.3 ± 0.1 and a k2max value of (3.1 ± 0.7)·104 m–1 s–1 for Cys-51 and a pKapp of 9.8 ± 0.1 and a k2max value of (2.6 ± 0.6)·104 m–1 s–1 for Cys-198 were determined (Fig. 1B). Altogether, the data support a pKapp value of both Cys-51 and Cys-198 in the reduced free wild-type enzyme close to 9.5.Direct Thiolate UV Absorbance of Reduced Wild-type, C51S, and C198S MsrAs—The thiolate absorbance of wild-type, C51S, and C198S MsrAs was monitored between pH 6 and 10. Analysis of the spectra and of the ϵ240 nm as a function of pH yielded monosigmoidal plots for all three MsrAs. Data fitted to pK values of 9.7, 9.8, and 9.7, associated with Δϵ240 nm of 3.1·104, 2.3·104, and 2.6·104 m–1·cm–1 for wild-type, C51S, and C198S MsrAs, respectively (Fig. 2). These pKapp values of Cys-51 and Cys-198 in the reduced free enzyme are in good agreement with those obtained with 2PDS.FIGURE 2pH dependence of the Cys thiol group absorbance properties of wild-type (•), C51S (Δ), and C198S (□) MsrAs. Molecular absorption coefficients at 240 nm were calculated from the absorbance spectra from 300 to 200 nm performed over a pH range of 7 to 10 in polybuffer B. Absorption coefficients were fitted to Equation 3 (solid line) (see also "Experimental Procedures").View Large Image Figure ViewerDownload Hi-res image Download (PPT)Kinetic Characterization with Identification of the Rate-limiting Step of the Mutated MsrAs at pH 8Steady-state catalytic constants of mutated MsrAs at positions 82, 94, and/or 134 were determined at pH 8, which is the optimum pH for the wild type (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). AcMetSONHMe was used instead of MetSO because MsrA displays a better affinity for AcMetSONHMe (9Boschi-Muller S. Olry A. Antoine M. Branlant G. Biochim. Biophys. Acta. 2005; 1703: 231-238Crossref PubMed Scopus (154) Google Scholar). As shown in Table 1, Y82F, Y134F, and E94Q MsrAs exhibited slight modifications of kcat compared with wild-type MsrA, with kcat values from 0.9 to 2.2 s–1 and a Km increase from 0.8 to 25 mm. In contrast, E94A, E94D, Y82F/Y134F, and Y82F/Y134F/E94Q MsrAs showed strongly decreased kcat values, from 1·10–3 to 2.5·10–1 s–1 and increased Km from 24 to 161 mm.TABLE 1Steady-state and reductase step kinetic parameters of wild-type and mutated MsrAs, with associated pKappSteady-stateaSteady-state parameters were deduced from nonlinear regression of initial rates to the Michaelis-Menten relationship (see "Experimental Procedures").Reductase stepkcatKmkmaxbKinetic parameters of the reductase step were obtained from nonlinear regression of kobs to Equation 7 (see "Experimental Procedures"), except for E94A and E94D MsrAs. For these two latter substituted MsrAs, kmax and KS values correspond to kcat and Km values determined under steady-state conditions (see "Results").KSbKinetic parameters of the reductase step were obtained from nonlinear regression of kobs to Equation 7 (see "Experimental Procedures"), except for E94A and E94D MsrAs. For these two latter substituted MsrAs, kmax and KS values correspond to kcat and Km values determined under steady-state conditions (see "Results").kmax optcKinetic parameters, kmax opt at optimum pH and pKapp values, were deduced from nonlinear regression of kobs to Equation 8 (see also Fig. 4).pKappcKinetic parameters, kmax opt at optimum pH and pKapp values, were deduced from nonlinear regression of kobs to Equation 8 (see also Fig. 4).s-1, pH 8.0mm, pH 8.0s-1, pH 8.0mm, pH 8.0s-1, optimal pHWild type3.7 ± 0.50.6 ± 0.2790 ± 1055 ± 2730 ± 105.7 ± 0.1E94A(1.5 ± 0.1)·10-2119 ± 27(1.5 ± 0.1)·10-2119 ± 27(2.0 ± 0.1)·10-27.5 ± 0.1E94D0.25 ± 0.03161 ± 420.25 ± 0.03161 ± 420.19 ± 0.016.7 ± 0.1E94Q0.88 ± 0.0725 ± 612.2 ± 0.3151 ± 828 ± 38.0 ± 0.1Y82F2.2 ± 0.13.7 ± 0.751 ± 172 ± 446 ± 17.6 ± 0.1Y134F1.5 ± 0.10.8 ± 0.2250 ± 870 ± 10380 ± 107.7 ± 0.1Y82F/Y134F(1.5 ± 0.1)·10-224 ± 8(3.4 ± 0.1)·10-226 ± 2(7.2 ± 0.7)·10-28.0 ± 0.1Y82F/Y134F/E94Q(1.0 ± 0.1)·10-362 ± 22(1.2 ± 0.1)·10-328 ± 7(1.1 ± 0.1)·10-29.5 ± 0.1a Steady-state parameters were deduced from nonlinear regression of initial rates to the Michaelis-Menten relationship (see "Experimental Procedures").b Kinetic parameters of the reductase step were obtained from nonlinear regression of kobs to Equation 7 (see "Experimental Procedures"), except for E94A and E94D MsrAs. For these two latter substituted MsrAs, kmax and KS values correspond to kcat and Km values determined under steady-state conditions (see "Results").c Kinetic parameters, kmax opt at optimum pH and pKapp values, were deduced from nonlinear regression of kobs to Equation 8 (see also Fig. 4). Open table in a new tab In the wild type, the rate of the reductase step is largely higher than the kcat value (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Therefore, to interpret the eventual kinetic consequences of the substitutions at positions 82, 94, and 134 at the level of the reductase step, it was first necessary to attain this rate. This was determined for E94Q, Y82F, Y134F, Y82F/Y134F, and Y82F/Y134F/E94Q MsrAs by following the variation of the Trp-53 fluorescence intensity under single turnover conditions, i.e. in the absence of reductant (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In that context, it was assumed that the reductase step of the mutated MsrAs is still rate-determining in the process leading to formation of the Msr disulfide bond, 4For each Glu-94-mutated MsrA, the rate constant of the reductase step was shown to be rate-determining in the two-step process leading to the formation of the disulfide bond by following the rate of AcMetNHMe and disulfide bond formation directly by acid-quenching of the reaction, followed by quantification of product and MsrA remaining free thiols (see "Results" for E94A and E94D MsrAs, data not shown for E94Q). For each Tyr-substituted MsrA, the rate of the reductase step was shown to be too fast at pH 8 to allow direct determination of the rate of AcMetNHMe and disulfide bond formation (data not shown). as previously shown for the wild type (1Antoine M. Boschi-Muller S. Branlant G. J. Biol. Chem. 2003; 278: 45352-45357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In the case of E94Q, Y82F, and Y134F MsrAs, formation of the disulfide bond led to an increase in the Trp-53 fluorescence emission similar to that described for the wild type, whereas a quenching of the fluorescence was observed for Y82F/Y134F and Y82F/Y134F/E94Q MsrAs.Structural factors responsible for this different behavior remain unknown. For all mutated MsrAs, the variation of the fluorescence signal in function of time is of monoexponential type whatever the AcMetSONHMe concentration. The kinetic parameter