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Antibiotic resistance in Gram-negative bacteria is a major health concern. It is principally observed due to the emergence of β-lactamase producers, which leads to the resistance against β-lactam antibiotics. However, their activity is inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Therefore researchers proposed that combination therapy with β-lactam antibiotics and β-lactamase inhibitors would provide a novel approach to overcoming antibiotic resistance in the future.
To first identify the kinetic effects of β-lactamase inhibitors scientists incubated β-lactamases in the presence of nitrocefin a chromogenic cephalosporin which changes absorbance upon cleavage from 380 nm to 500 nm. The rate of nitrocefin hydrolysis was measured in the presence and absence of different inhibitors and the results were plotted in Lineweaver-Burk plots.
To study the effectiveness of combination therapy researchers transfected pQE-2-CTX-M-15 plasmids which encodes for the β-lactamase into E. Coli. The cells were then cultured in the presence of different antibiotics or combination therapy and the minimal inhibitory concentration for each condition was measured. The results are shown in Table 1.
Table 1: MIC measured as the lowest dose to inhibit bacterial growth by 50% for cultures with β-lactam antibiotics and combination therapy with β-lactamase inhibitors.
Antibiotic + Inhibitor | MIC (μg/mL) w/pQE-2-CTX-M-15 | MIC (μg/mL) w/null Plasmid |
Ampicillin | >1025 | 2 |
Ampicillin + Clavulanic Acid | 520 | 2 |
Cefoxitin | 16 | 2 |
Cefoxitin + Clavulanic Acid | 2 | 1 |
Cefepime | 128 | 0.25 |
Cefepime + Clavulanic Acid | 16 | 0.02 |
Adapted From: Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor Faheem M, Rehman MT, Danishuddin M, Khan AU (2013) Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β Lactamase Inhibitor. PLOS ONE 8(2): e56926. https://doi.org/10.1371/journal.pone.0056926
β-lactamase enzymes require what additional chemical compound to catalyze the breakdown of β-lactam antibiotics?
Antibiotic resistance in Gram-negative bacteria is a major health concern. It is principally observed due to the emergence of β-lactamase producers, which leads to the resistance against β-lactam antibiotics. However, their activity is inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Therefore researchers proposed that combination therapy with β-lactam antibiotics and β-lactamase inhibitors would provide a novel approach to overcoming antibiotic resistance in the future.
To first identify the kinetic effects of β-lactamase inhibitors scientists incubated β-lactamases in the presence of nitrocefin a chromogenic cephalosporin which changes absorbance upon cleavage from 380 nm to 500 nm. The rate of nitrocefin hydrolysis was measured in the presence and absence of different inhibitors and the results were plotted in Lineweaver-Burk plots.
To study the effectiveness of combination therapy researchers transfected pQE-2-CTX-M-15 plasmids which encodes for the β-lactamase into E. Coli. The cells were then cultured in the presence of different antibiotics or combination therapy and the minimal inhibitory concentration for each condition was measured. The results are shown in Table 1.
Table 1: MIC measured as the lowest dose to inhibit bacterial growth by 50% for cultures with β-lactam antibiotics and combination therapy with β-lactamase inhibitors.
Antibiotic + Inhibitor | MIC (μg/mL) w/pQE-2-CTX-M-15 | MIC (μg/mL) w/null Plasmid |
Ampicillin | >1025 | 2 |
Ampicillin + Clavulanic Acid | 520 | 2 |
Cefoxitin | 16 | 2 |
Cefoxitin + Clavulanic Acid | 2 | 1 |
Cefepime | 128 | 0.25 |
Cefepime + Clavulanic Acid | 16 | 0.02 |
Adapted From: Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor Faheem M, Rehman MT, Danishuddin M, Khan AU (2013) Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β Lactamase Inhibitor. PLOS ONE 8(2): e56926. https://doi.org/10.1371/journal.pone.0056926
What is the difference in energy between the photons emitted by uncleaved nitrocefin and cleaved nitrocefin? (Note: h = 6.63 x 10-23).
Antibiotic resistance in Gram-negative bacteria is a major health concern. It is principally observed due to the emergence of β-lactamase producers, which leads to the resistance against β-lactam antibiotics. However, their activity is inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Therefore researchers proposed that combination therapy with β-lactam antibiotics and β-lactamase inhibitors would provide a novel approach to overcoming antibiotic resistance in the future.
To first identify the kinetic effects of β-lactamase inhibitors scientists incubated β-lactamases in the presence of nitrocefin a chromogenic cephalosporin which changes absorbance upon cleavage from 380 nm to 500 nm. The rate of nitrocefin hydrolysis was measured in the presence and absence of different inhibitors and the results were plotted in Lineweaver-Burk plots.
To study the effectiveness of combination therapy researchers transfected pQE-2-CTX-M-15 plasmids which encodes for the β-lactamase into E. Coli. The cells were then cultured in the presence of different antibiotics or combination therapy and the minimal inhibitory concentration for each condition was measured. The results are shown in Table 1.
Table 1: MIC measured as the lowest dose to inhibit bacterial growth by 50% for cultures with β-lactam antibiotics and combination therapy with β-lactamase inhibitors.
Antibiotic + Inhibitor | MIC (μg/mL) w/pQE-2-CTX-M-15 | MIC (μg/mL) w/null Plasmid |
Ampicillin | >1025 | 2 |
Ampicillin + Clavulanic Acid | 520 | 2 |
Cefoxitin | 16 | 2 |
Cefoxitin + Clavulanic Acid | 2 | 1 |
Cefepime | 128 | 0.25 |
Cefepime + Clavulanic Acid | 16 | 0.02 |
Adapted From: Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor Faheem M, Rehman MT, Danishuddin M, Khan AU (2013) Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β Lactamase Inhibitor. PLOS ONE 8(2): e56926. https://doi.org/10.1371/journal.pone.0056926
Clavulanic acid inhibits β-lactamase activity by:
Antibiotic resistance in Gram-negative bacteria is a major health concern. It is principally observed due to the emergence of β-lactamase producers, which leads to the resistance against β-lactam antibiotics. However, their activity is inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Therefore researchers proposed that combination therapy with β-lactam antibiotics and β-lactamase inhibitors would provide a novel approach to overcoming antibiotic resistance in the future.
To first identify the kinetic effects of β-lactamase inhibitors scientists incubated β-lactamases in the presence of nitrocefin a chromogenic cephalosporin which changes absorbance upon cleavage from 380 nm to 500 nm. The rate of nitrocefin hydrolysis was measured in the presence and absence of different inhibitors and the results were plotted in Lineweaver-Burk plots.
To study the effectiveness of combination therapy researchers transfected pQE-2-CTX-M-15 plasmids which encodes for the β-lactamase into E. Coli. The cells were then cultured in the presence of different antibiotics or combination therapy and the minimal inhibitory concentration for each condition was measured. The results are shown in Table 1.
Table 1: MIC measured as the lowest dose to inhibit bacterial growth by 50% for cultures with β-lactam antibiotics and combination therapy with β-lactamase inhibitors.
Antibiotic + Inhibitor | MIC (μg/mL) w/pQE-2-CTX-M-15 | MIC (μg/mL) w/null Plasmid |
Ampicillin | >1025 | 2 |
Ampicillin + Clavulanic Acid | 520 | 2 |
Cefoxitin | 16 | 2 |
Cefoxitin + Clavulanic Acid | 2 | 1 |
Cefepime | 128 | 0.25 |
Cefepime + Clavulanic Acid | 16 | 0.02 |
Adapted From: Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor Faheem M, Rehman MT, Danishuddin M, Khan AU (2013) Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β Lactamase Inhibitor. PLOS ONE 8(2): e56926. https://doi.org/10.1371/journal.pone.0056926
β-lactamase would most likely catalyze a reaction which of the following substrate molecules?
Antibiotic resistance in Gram-negative bacteria is a major health concern. It is principally observed due to the emergence of β-lactamase producers, which leads to the resistance against β-lactam antibiotics. However, their activity is inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Therefore researchers proposed that combination therapy with β-lactam antibiotics and β-lactamase inhibitors would provide a novel approach to overcoming antibiotic resistance in the future.
To first identify the kinetic effects of β-lactamase inhibitors scientists incubated β-lactamases in the presence of nitrocefin a chromogenic cephalosporin which changes absorbance upon cleavage from 380 nm to 500 nm. The rate of nitrocefin hydrolysis was measured in the presence and absence of different inhibitors and the results were plotted in Lineweaver-Burk plots.
To study the effectiveness of combination therapy researchers transfected pQE-2-CTX-M-15 plasmids which encodes for the β-lactamase into E. Coli. The cells were then cultured in the presence of different antibiotics or combination therapy and the minimal inhibitory concentration for each condition was measured. The results are shown in Table 1.
Table 1: MIC measured as the lowest dose to inhibit bacterial growth by 50% for cultures with β-lactam antibiotics and combination therapy with β-lactamase inhibitors.
Antibiotic + Inhibitor | MIC (μg/mL) w/pQE-2-CTX-M-15 | MIC (μg/mL) w/null Plasmid |
Ampicillin | >1025 | 2 |
Ampicillin + Clavulanic Acid | 520 | 2 |
Cefoxitin | 16 | 2 |
Cefoxitin + Clavulanic Acid | 2 | 1 |
Cefepime | 128 | 0.25 |
Cefepime + Clavulanic Acid | 16 | 0.02 |
Adapted From: Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor Faheem M, Rehman MT, Danishuddin M, Khan AU (2013) Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β Lactamase Inhibitor. PLOS ONE 8(2): e56926. https://doi.org/10.1371/journal.pone.0056926
What is the Km and Vmax for the most effective inhibitor?
Antibiotic resistance in Gram-negative bacteria is a major health concern. It is principally observed due to the emergence of β-lactamase producers, which leads to the resistance against β-lactam antibiotics. However, their activity is inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Therefore researchers proposed that combination therapy with β-lactam antibiotics and β-lactamase inhibitors would provide a novel approach to overcoming antibiotic resistance in the future.
To first identify the kinetic effects of β-lactamase inhibitors scientists incubated β-lactamases in the presence of nitrocefin a chromogenic cephalosporin which changes absorbance upon cleavage from 380 nm to 500 nm. The rate of nitrocefin hydrolysis was measured in the presence and absence of different inhibitors and the results were plotted in Lineweaver-Burk plots.
To study the effectiveness of combination therapy researchers transfected pQE-2-CTX-M-15 plasmids which encodes for the β-lactamase into E. Coli. The cells were then cultured in the presence of different antibiotics or combination therapy and the minimal inhibitory concentration for each condition was measured. The results are shown in Table 1.
Table 1: MIC measured as the lowest dose to inhibit bacterial growth by 50% for cultures with β-lactam antibiotics and combination therapy with β-lactamase inhibitors.
Antibiotic + Inhibitor | MIC (μg/mL) w/pQE-2-CTX-M-15 | MIC (μg/mL) w/null Plasmid |
Ampicillin | >1025 | 2 |
Ampicillin + Clavulanic Acid | 520 | 2 |
Cefoxitin | 16 | 2 |
Cefoxitin + Clavulanic Acid | 2 | 1 |
Cefepime | 128 | 0.25 |
Cefepime + Clavulanic Acid | 16 | 0.02 |
Adapted From: Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor Faheem M, Rehman MT, Danishuddin M, Khan AU (2013) Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β Lactamase Inhibitor. PLOS ONE 8(2): e56926. https://doi.org/10.1371/journal.pone.0056926
Which of the following would be LEAST effective in treating a gram-negative bacterial infection that has the pQE-2-CTX-M-15 plasmid?
Hemoproteins serve multiple roles in normal cellular functioning and significant homology exists between various hemoproteins. These structure-function similarities, as well as disparities, can be drawn between the two heme proteins, chloroperoxidase (CPO) and cytochrome P450 (CYP). The former is a highly stable glycosylated extracellular acidic protein with a constrained and polar active site. The latter is a relatively sensitive microsomal membranous protein showing little post-translational modifications, but with a larger hydrophobic active site. The most important common structural element is the proximal thiolate ligand, bound to the central iron of heme (protoporphyrin IX). The formal charge on iron at the resting state is 3+ and may change based on the distal ligand and microenvironment. Both CPO and CYPs are known for a relative lack of specificity in substrate preferences. CPO is a classical peroxygenase while CYP is a typical monooxygenase that requires a ternary mixture of molecular oxygen, another enzyme called cytochrome P450 reductase (CPR), and NADPH.
Recent observations demonstrate that CPO and CYP might not go through the typical enzyme-substrate binding at a unique active site of the enzyme. As a result, they may not be defined by the classical Michaelis-Menten kinetics. To see if this was the case researchers incubated CPO with varying concentrations of different substrates and plotted the data (Figure 1).
Figure 1: Kinetics of CPO catalyzed peroxidation of ABTS obtained by varying the ABTS concentration, at constant peroxide (left). Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD). Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 200 mM
The active site of CYP would contain which of the following amino acids?
Hemoproteins serve multiple roles in normal cellular functioning and significant homology exists between various hemoproteins. These structure-function similarities, as well as disparities, can be drawn between the two heme proteins, chloroperoxidase (CPO) and cytochrome P450 (CYP). The former is a highly stable glycosylated extracellular acidic protein with a constrained and polar active site. The latter is a relatively sensitive microsomal membranous protein showing little post-translational modifications, but with a larger hydrophobic active site. The most important common structural element is the proximal thiolate ligand, bound to the central iron of heme (protoporphyrin IX). The formal charge on iron at the resting state is 3+ and may change based on the distal ligand and microenvironment. Both CPO and CYPs are known for a relative lack of specificity in substrate preferences. CPO is a classical peroxygenase while CYP is a typical monooxygenase that requires a ternary mixture of molecular oxygen, another enzyme called cytochrome P450 reductase (CPR), and NADPH.
Recent observations demonstrate that CPO and CYP might not go through the typical enzyme-substrate binding at a unique active site of the enzyme. As a result, they may not be defined by the classical Michaelis-Menten kinetics. To see if this was the case researchers incubated CPO with varying concentrations of different substrates and plotted the data (Figure 1).
Figure 1: Kinetics of CPO catalyzed peroxidation of ABTS obtained by varying the ABTS concentration, at constant peroxide (left). Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD). Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 200 mM
What is the Kcat of ABTS by CPO when there is 1 mM Peroxide concentration?
Hemoproteins serve multiple roles in normal cellular functioning and significant homology exists between various hemoproteins. These structure-function similarities, as well as disparities, can be drawn between the two heme proteins, chloroperoxidase (CPO) and cytochrome P450 (CYP). The former is a highly stable glycosylated extracellular acidic protein with a constrained and polar active site. The latter is a relatively sensitive microsomal membranous protein showing little post-translational modifications, but with a larger hydrophobic active site. The most important common structural element is the proximal thiolate ligand, bound to the central iron of heme (protoporphyrin IX). The formal charge on iron at the resting state is 3+ and may change based on the distal ligand and microenvironment. Both CPO and CYPs are known for a relative lack of specificity in substrate preferences. CPO is a classical peroxygenase while CYP is a typical monooxygenase that requires a ternary mixture of molecular oxygen, another enzyme called cytochrome P450 reductase (CPR), and NADPH.
Recent observations demonstrate that CPO and CYP might not go through the typical enzyme-substrate binding at a unique active site of the enzyme. As a result, they may not be defined by the classical Michaelis-Menten kinetics. To see if this was the case researchers incubated CPO with varying concentrations of different substrates and plotted the data (Figure 1).
Figure 1: Kinetics of CPO catalyzed peroxidation of ABTS obtained by varying the ABTS concentration, at constant peroxide (left). Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD). Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 200 mM
The thiolate is bound to heme via which type of bond?
Hemoproteins serve multiple roles in normal cellular functioning and significant homology exists between various hemoproteins. These structure-function similarities, as well as disparities, can be drawn between the two heme proteins, chloroperoxidase (CPO) and cytochrome P450 (CYP). The former is a highly stable glycosylated extracellular acidic protein with a constrained and polar active site. The latter is a relatively sensitive microsomal membranous protein showing little post-translational modifications, but with a larger hydrophobic active site. The most important common structural element is the proximal thiolate ligand, bound to the central iron of heme (protoporphyrin IX). The formal charge on iron at the resting state is 3+ and may change based on the distal ligand and microenvironment. Both CPO and CYPs are known for a relative lack of specificity in substrate preferences. CPO is a classical peroxygenase while CYP is a typical monooxygenase that requires a ternary mixture of molecular oxygen, another enzyme called cytochrome P450 reductase (CPR), and NADPH.
Recent observations demonstrate that CPO and CYP might not go through the typical enzyme-substrate binding at a unique active site of the enzyme. As a result, they may not be defined by the classical Michaelis-Menten kinetics. To see if this was the case researchers incubated CPO with varying concentrations of different substrates and plotted the data (Figure 1).
Figure 1: Kinetics of CPO catalyzed peroxidation of ABTS obtained by varying the ABTS concentration, at constant peroxide (left). Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD). Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 200 mM
Adapted from: Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment Manoj KM, Baburaj A, Ephraim B, Pappachan F, Maviliparambathu PP, et al. (2010) Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment. PLOS ONE 5(5): e10601. https://doi.org/10.1371/journal.pone.0010601
Which of the following is a component of the CYP and CPO cofactor?
Hemoproteins serve multiple roles in normal cellular functioning and significant homology exists between various hemoproteins. These structure-function similarities, as well as disparities, can be drawn between the two heme proteins, chloroperoxidase (CPO) and cytochrome P450 (CYP). The former is a highly stable glycosylated extracellular acidic protein with a constrained and polar active site. The latter is a relatively sensitive microsomal membranous protein showing little post-translational modifications, but with a larger hydrophobic active site. The most important common structural element is the proximal thiolate ligand, bound to the central iron of heme (protoporphyrin IX). The formal charge on iron at the resting state is 3+ and may change based on the distal ligand and microenvironment. Both CPO and CYPs are known for a relative lack of specificity in substrate preferences. CPO is a classical peroxygenase while CYP is a typical monooxygenase that requires a ternary mixture of molecular oxygen, another enzyme called cytochrome P450 reductase (CPR), and NADPH.
Recent observations demonstrate that CPO and CYP might not go through the typical enzyme-substrate binding at a unique active site of the enzyme. As a result, they may not be defined by the classical Michaelis-Menten kinetics. To see if this was the case researchers incubated CPO with varying concentrations of different substrates and plotted the data (Figure 1).
Figure 1: Kinetics of CPO catalyzed peroxidation of ABTS obtained by varying the ABTS concentration, at constant peroxide (left). Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD). Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 200 mM
Adapted from: Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment Manoj KM, Baburaj A, Ephraim B, Pappachan F, Maviliparambathu PP, et al. (2010) Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment. PLOS ONE 5(5): e10601. https://doi.org/10.1371/journal.pone.0010601
The experiment used a phosphate buffer to ensure that the pH was maintained at a pH = 3.5 throughout. At this pH what was the concentration of PO43- in the solution? (The pKa values for a phosphate buffer are as follows: pKa1 =2.5, pKa2 = 7.25 pKa3 = 12.5)