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Jean-pierre Samama - One of the best experts on this subject based on the ideXlab platform.
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Mass spectral kinetic study of Acylation and deAcylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant.
Biochemistry, 1995Co-Authors: Isabelle Saves, Odile Burlet-schiltz, Laurent Maveyraud, Jean-pierre Samama, Jean-claude Promé, Jean-michel MassonAbstract:The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the Acylation and deAcylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deAcylation for penicillin hydrolysis, but Acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deAcylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased Acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site.
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Electrostatic analysis of TEM1 β-lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations
Structure (London England : 1993), 1995Co-Authors: Peter Swarén, Laurent Maveyraud, Jean-michel Masson, Valérie Guillet, Lionel Mourey, Jean-pierre SamamaAbstract:Abstract Background: Escherichia coli TEM1 is a penicillinase and belongs to class A β -lactamases. Its naturally occurring mutants are responsible for bacterial resistance to β -lactamin-based antibiotics. X-ray structure determinations show that all class A β -lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the Acylation and deAcylation steps are and how they contribute to the efficiency of these penicillinases. Results Electrostatic analysis of the 1.8 a resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl–enzyme complexes, showed that substrate binding induces an upward shift in the pK a of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote Acylation. In the acyl–enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the ester-carbonyl carbon of the acyl–enzyme complex, in an electrostatic potential gradient amounting to 30 kTe −1 a −1 . Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deAcylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. Conclusion In the wild-type enzyme, the very high rates of Acylation and deAcylation of class A β -lactamases arise from an optimal chemical setup in which the Acylation reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, Acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.
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Electrostatic analysis of TEM1 -lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations
Structure, 1995Co-Authors: Peter Swarén, Laurent Maveyraud, Jean-michel Masson, Valérie Guillet, Lionel Mourey, Jean-pierre SamamaAbstract:Background: Escherichia coli TEM1 is a penicillinase and belongs to class A-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to 3-lactamin-based antibiotics. X-ray structure determina-tions show that all class A-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the Acylation and deAcylation steps are and how they contribute to the efficiency of these penicillinases. Results: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote Acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the ester-carbonyl carbon of the acyl-enzyme complex, in an elec-trostatic potential gradient amounting to 30 kTe-' A-'. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deAcylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-directed mutants that affect the protona-tion state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. Conclusions: In the wild-type enzyme, the very high rates of Acylation and deAcylation of class A P-lactamases arise from an optimal chemical setup in which the acyla-tion reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, Acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.
Paul A.g. Butler - One of the best experts on this subject based on the ideXlab platform.
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Lamb pregastric lipase catalysed hydrolysis of 1,2,3-tri[(cis)-9-octadecenoyl]glycerol: temperature, pH and solvent isotope effects
Journal of Molecular Catalysis A: Chemical, 1995Co-Authors: Charmian J. O'connor, Andrew D. Mackenzie, Richard H. Barton, Paul A.g. ButlerAbstract:Abstract Lamb pregastric lipase, extracted from the tongue and epiglottal region of lamb, has been partially purified and used to catalyse the hydrolysis of (9–10 3 H) (1,2,3-tris-[( cis )-9-octadecenoyl]glycerol) over the pH range 5.50–7.50 and temperature range 20.0–40.0°C. Michaelis-Menten plots were constructed for each reaction condition, and allowed evaluation of K m and k cat . The values of k cat have been fitted to a three-dimensional activity profile. The optimum pH for reactivity was 6.3 ± 0.3 and the optimum temperature 32 ± 3°C. Under optimum conditions the values of k cat and K m were 0.073 μmol · min −1 · mg −1 and 9 mM, respectively. The reaction has also been studied in D 2 O as solvent at pD = 6.50 and T = 30.0°C. The kinetic isotope effects were 1.46 and 1.31 for the rate determining Acylation and deAcylation steps, respectively. The activity of the enzyme was inhibited by the presence of the bile salt, sodium taurocholate.
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Bile-salt-stimulated human milk lipase catalysed hydrolysis of 1,2,3-tri[(cis)-9-octadecenoyl] glycerol : solvent isotope effect
Journal of Molecular Catalysis, 1994Co-Authors: Charmian J. O'connor, Douglas R. Cleverly, Paul A.g. ButlerAbstract:Abstract Bile salt stimulated lipase from human milk has been used to catalyse the hydrolysis of (9–10 3 H) (1,2,3-tri[( cis )-9-octadecenoyl]glycerol) in emulsion media containing buffer dissolvedin H 2 O and D 2 O. The reactions were carried out in the presence of 20 mM taurocholate at pH = pD = 7.5 and 37.5°C. Analysis of the data gave values of V max equal to 194 and 61.8 Bq·s −1 , and of K m equal to 3.48 and 2.91 mM, for reactions in H 2 O and D 2 O, respectively. The solvent isotope effects for Acylation and deAcylation were equal to 2.63 and 3.14, respectively. A mechanism has been proposed which explains the observed effects and takes into account the known amino acid sequence of the active site and the residues involved in catalysis.
Jean-michel Masson - One of the best experts on this subject based on the ideXlab platform.
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Mass spectral kinetic study of Acylation and deAcylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant.
Biochemistry, 1995Co-Authors: Isabelle Saves, Odile Burlet-schiltz, Laurent Maveyraud, Jean-pierre Samama, Jean-claude Promé, Jean-michel MassonAbstract:The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the Acylation and deAcylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deAcylation for penicillin hydrolysis, but Acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deAcylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased Acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site.
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Electrostatic analysis of TEM1 β-lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations
Structure (London England : 1993), 1995Co-Authors: Peter Swarén, Laurent Maveyraud, Jean-michel Masson, Valérie Guillet, Lionel Mourey, Jean-pierre SamamaAbstract:Abstract Background: Escherichia coli TEM1 is a penicillinase and belongs to class A β -lactamases. Its naturally occurring mutants are responsible for bacterial resistance to β -lactamin-based antibiotics. X-ray structure determinations show that all class A β -lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the Acylation and deAcylation steps are and how they contribute to the efficiency of these penicillinases. Results Electrostatic analysis of the 1.8 a resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl–enzyme complexes, showed that substrate binding induces an upward shift in the pK a of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote Acylation. In the acyl–enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the ester-carbonyl carbon of the acyl–enzyme complex, in an electrostatic potential gradient amounting to 30 kTe −1 a −1 . Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deAcylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. Conclusion In the wild-type enzyme, the very high rates of Acylation and deAcylation of class A β -lactamases arise from an optimal chemical setup in which the Acylation reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, Acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.
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Electrostatic analysis of TEM1 -lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations
Structure, 1995Co-Authors: Peter Swarén, Laurent Maveyraud, Jean-michel Masson, Valérie Guillet, Lionel Mourey, Jean-pierre SamamaAbstract:Background: Escherichia coli TEM1 is a penicillinase and belongs to class A-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to 3-lactamin-based antibiotics. X-ray structure determina-tions show that all class A-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the Acylation and deAcylation steps are and how they contribute to the efficiency of these penicillinases. Results: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote Acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the ester-carbonyl carbon of the acyl-enzyme complex, in an elec-trostatic potential gradient amounting to 30 kTe-' A-'. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deAcylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-directed mutants that affect the protona-tion state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. Conclusions: In the wild-type enzyme, the very high rates of Acylation and deAcylation of class A P-lactamases arise from an optimal chemical setup in which the acyla-tion reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, Acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.
Anthony L. Fink - One of the best experts on this subject based on the ideXlab platform.
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Lysine-73 is involved in the Acylation and deAcylation of beta-lactamase.
Biochemistry, 2000Co-Authors: Eric J. Lietz, Heather Truher, Debra Kahn, Mark J. Hokenson, Anthony L. FinkAbstract:Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K(m)), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K(m)) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K(m) vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K(m) at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deAcylation. FTIR analysis was used to determine whether Acylation or deAcylation was rate-limiting. In general, Acylation was the rate-limiting step for cephalosporin substrates, whereas deAcylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the Acylation and deAcylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.
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Site-directed mutagenesis of .beta.-lactamase leading to accumulation of a catalytic intermediate
Biochemistry, 1991Co-Authors: Walter A. Escobar, Anthony K. Tan, Anthony L. FinkAbstract:Site-specific mutation of Glu-166 to Ala in beta-lactamase causes a millionfold reduction in catalytic activity toward both penicillin and cephalosporin substrates and results in the stoichiometric accumulation of a normally transient acyl-enzyme intermediate. Kinetic analysis indicated that substitution of Glu-166 by Ala leads to negligible effect on the Acylation half of the reaction but effectively eliminates the deAcylation reaction. Such differential effects on the rates of formation and breakdown of an enzyme-substrate intermediate have not been previously reported. Thus, unlike the situation for most transfer enzymes, e.g., the serine proteases, Acylation and deAcylation in beta-lactamase catalysis are not "mirror" images and must involve different mechanisms. The results suggest an explanation for the different catalytic activities between the beta-lactamases and the penicillin-binding proteins involved in bacterial cell-wall synthesis.
Laurent Maveyraud - One of the best experts on this subject based on the ideXlab platform.
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Mass spectral kinetic study of Acylation and deAcylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant.
Biochemistry, 1995Co-Authors: Isabelle Saves, Odile Burlet-schiltz, Laurent Maveyraud, Jean-pierre Samama, Jean-claude Promé, Jean-michel MassonAbstract:The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the Acylation and deAcylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deAcylation for penicillin hydrolysis, but Acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deAcylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased Acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site.
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Electrostatic analysis of TEM1 β-lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations
Structure (London England : 1993), 1995Co-Authors: Peter Swarén, Laurent Maveyraud, Jean-michel Masson, Valérie Guillet, Lionel Mourey, Jean-pierre SamamaAbstract:Abstract Background: Escherichia coli TEM1 is a penicillinase and belongs to class A β -lactamases. Its naturally occurring mutants are responsible for bacterial resistance to β -lactamin-based antibiotics. X-ray structure determinations show that all class A β -lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the Acylation and deAcylation steps are and how they contribute to the efficiency of these penicillinases. Results Electrostatic analysis of the 1.8 a resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl–enzyme complexes, showed that substrate binding induces an upward shift in the pK a of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote Acylation. In the acyl–enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the ester-carbonyl carbon of the acyl–enzyme complex, in an electrostatic potential gradient amounting to 30 kTe −1 a −1 . Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deAcylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. Conclusion In the wild-type enzyme, the very high rates of Acylation and deAcylation of class A β -lactamases arise from an optimal chemical setup in which the Acylation reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, Acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.
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Electrostatic analysis of TEM1 -lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations
Structure, 1995Co-Authors: Peter Swarén, Laurent Maveyraud, Jean-michel Masson, Valérie Guillet, Lionel Mourey, Jean-pierre SamamaAbstract:Background: Escherichia coli TEM1 is a penicillinase and belongs to class A-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to 3-lactamin-based antibiotics. X-ray structure determina-tions show that all class A-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the Acylation and deAcylation steps are and how they contribute to the efficiency of these penicillinases. Results: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote Acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the ester-carbonyl carbon of the acyl-enzyme complex, in an elec-trostatic potential gradient amounting to 30 kTe-' A-'. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deAcylation rate is related to the magnitude of the gradient. The kinetic behaviour of site-directed mutants that affect the protona-tion state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. Conclusions: In the wild-type enzyme, the very high rates of Acylation and deAcylation of class A P-lactamases arise from an optimal chemical setup in which the acyla-tion reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, Acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.
