Steady-State Kinetic Analysis of Phosphotransacetylase from Methanosarcina thermophila
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《细菌学杂志》
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500
ABSTRACT
Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. A steady-state kinetic analysis of the phosphotransacetylase from Methanosarcina thermophila indicated that there is a ternary complex kinetic mechanism rather than a ping-pong kinetic mechanism. Additionally, inhibition patterns of products and a nonreactive substrate analog suggested that the substrates bind to the enzyme in a random order. Dynamic light scattering revealed that the enzyme is dimeric in solution.
TEXT
Together with acetate kinase (equation 2), phosphotransacetylase (Pta) plays an essential role (equation 1) in the conversion of acetyl coenzyme A (acetyl-CoA) to acetate and in the synthesis of ATP in fermentative anaerobes belonging to the domain Bacteria. Acetate kinase and Pta also activate acetate to acetyl-CoA (reverse of equations 1 and 2) for conversion to methane and carbon dioxide in the energy-yielding metabolism of Methanosarcina species belonging to the domain Archaea.
(1)
(2)
Pta was first purified from a fermentative organism, Clostridium kluyveri, in the 1950s (13). Early kinetic analyses of the Ptas from C. kluyveri and Veillonella alcalescens belonging to the domain Bacteria are consistent with the presence of a ternary complex kinetic mechanism (2, 6, 10). Mechanistic analyses of the enzyme were abandoned until cloning and heterologous expression of Pta from Methanosarcina thermophila, a methane-producing organism belonging to the domain Archaea, which allowed application of modern biochemical techniques (7). The recently published crystal structure of M. thermophila Pta, along with kinetic analyses of site-specific replacement variants (3, 8, 11), have made this enzyme the preferred model for elucidation of the catalytic mechanism of Pta. As Ptas from diverse fermentative microbes belonging to the domain Bacteria exhibit high levels of sequence identity (4), an understanding of the M. thermophila Pta can be extrapolated to all Ptas. The steady-state kinetic analysis of M. thermophila Pta presented here suggests that the kinetic mechanism proceeds through random formation of a ternary complex. Our results provide a kinetic foundation essential for interpreting structural information for the M. thermophila Pta (4, 8) in order to elucidate the catalytic mechanism.
The Pta from M. thermophila was heterologously expressed and purified as described previously (5), and the preparation appeared to be homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The homogeneity and approximate hydrodynamic radius of Pta were examined by dynamic light scattering (DLS) using a DynaPro-MS800 molecular sizing instrument (Protein Solutions, Lakewood, NJ) as follows. A 40-μl aliquot of Pta (2.5 mg/ml) in 25 mM Tris-HCl (pH 7.2) containing 180 mM KCl was centrifuged (10,000 x g, 10 min), and an aliquot was loaded into a 12-μl quartz cuvette. The hydrodynamic radius, molecular weight, and size distribution were determined by the means of at least 10 DLS measurements. Data analysis was performed using Dynamics 5.0 (Protein Solutions, Lakewood, NJ). A sample DLS data set is shown in Fig. 1. Although the enzyme was initially reported to exist in solution as a monomer (7), Pta was found to have a hydrodynamic radius of 3.7 ± 0.1 nm, corresponding to a molecular mass of 71 ± 3 kDa, which is twice the calculated molecular mass of a Pta monomer. The observed molecular mass indicated that Pta exists in solution as a dimer, which is consistent with the dimeric states observed for the crystal structures of Ptas from M. thermophila and Streptococcus pyogenes (4, 14).
The rates for both the forward (acetyl-CoA-forming) and reverse (acetyl phosphate-forming) directions of the reaction catalyzed by Pta were measured at 25°C by monitoring the change in absorbance at 233 nm concomitant with formation or hydrolysis of the thioester bond of acetyl-CoA ( = 4,360 M–1), using a 0.1-cm-path-length quartz cuvette in a Hewlett-Packard 8452A diode array spectrophotometer. The standard reaction mixture (200 μl) contained 50 mM Tris-HCl (pH 7.2), 20 mM NH4Cl, 20 mM KCl, 2 mM dithiothreitol, the appropriate substrate for the experiment, and a concentration of Pta sufficient to yield a linear rate over at least 2 min (usually 0.05 μg/ml). Reactions were initiated by addition of the second substrate. All components were maintained on ice and warmed to 25°C immediately prior to initiation of the reaction.
The initial velocity patterns of the two-substrate, two-product reaction catalyzed by Pta were investigated in order to differentiate between a ternary complex kinetic mechanism and a ping-pong kinetic mechanism. For each direction of the reaction catalyzed by Pta, the initial velocity of the reaction was measured by using a matrix of five different concentrations of each substrate. Rates were measured using the standard activity assay, and each reaction was initiated by addition of the varied substrate. Data were expressed as double-reciprocal plots and fitted globally using Grafit 5.0 (9) to equation 3 describing the pattern for a ternary complex kinetic mechanism (1):
(3)
where V is the maximal velocity, A and B are the concentrations of the varied and fixed substrates, respectively, KA and KB are the Michaelis constants for substrates A and B, respectively, and KD(A) is the dissociation constant for the varied substrate.
For both directions, the data yielded sets of intersecting lines fitted to equation 1 (Fig. 2). For the forward direction, Michaelis constants of 186 ± 6 and 65 ± 7 μM were obtained for acetyl phosphate and CoA, respectively, and the kcat was 5,190 ± 30 s–1. For the reverse direction, Michaelis constants of 96 ± 13 and 742 ± 86 μM were obtained for acetyl-CoA and phosphate, respectively, and the kcat was 1,500 ± 30 s–1. The initial velocity patterns were similar to those observed for the Ptas from V. alcalescens and C. kluyveri (2, 10) and are consistent with a kinetic mechanism that proceeds via formation of a ternary complex between Pta and both substrates prior to any chemical step, rather than via a ping-pong mechanism.
The product inhibition patterns of the reaction catalyzed by Pta were analyzed to determine if substrate binding and product release are random or ordered. Inhibition of the forward (acetyl-CoA-forming) reaction catalyzed by Pta by the product inhibitors, acetyl-CoA and inorganic phosphate, was analyzed with respect to various concentrations of the substrates, CoA and acetyl phosphate, using the standard activity assay. All four product-substrate pairs were analyzed at saturating and subsaturating conditions using a matrix of five concentrations of substrates and inhibitors for each experiment. Data were expressed as double-reciprocal plots and analyzed to determine the nature of the inhibition. Data were fitted using Grafit 5.0 (9) to equations describing competitive (equation 4) or noncompetitive (equation 5) inhibition using two-dimensional least-squares analysis (12).
(4)
(5)
where Km is the Michaelis constant for the substrate, S is the concentration of the substrate, I is the concentration of the inhibitor, and Ki is the inhibition constant for the product inhibitor.
Phosphate was a competitive inhibitor versus acetyl phosphate when CoA was at saturating (600 μM) or subsaturating (60 μM) levels (Fig. 3A and B). Phosphate was a noncompetitive inhibitor versus CoA when acetyl phosphate was at a subsaturating level (150 μM) (Fig. 3C), but it did not inhibit versus CoA when acetyl phosphate was at a saturating level (4mM). Acetyl-CoA was a competitive inhibitor versus CoA when acetyl phosphate was at subsaturating levels (Fig. 3D), but it did not inhibit versus CoA when acetyl phosphate was at saturating levels. Similarly, acetyl-CoA was a competitive inhibitor versus acetyl phosphate when CoA was at subsaturating levels (Fig. 3E), but it did not inhibit versus acetyl phosphate when CoA was at saturating levels. This pattern of inhibition is diagnostic for a kinetic mechanism that proceeds through formation of a ternary complex in which the substrates can bind to the enzyme in random order (1, 12).
The inhibition patterns of the nonreactive CoA analogue desulfo-CoA were analyzed with respect to CoA and acetyl phosphate, and the results further supported the random binding suggested by the product inhibition patterns. Data were expressed as double-reciprocal plots and fitted to equations 4 and 5 describing competitive and noncompetitive inhibition, respectively. Desulfo-CoA was a competitive inhibitor with respect to CoA (Fig. 4A), with a Ki of 1.3 ± 0.1 μM, confirming a previous report (3). Desulfo-CoA was a noncompetitive inhibitor with respect to acetyl phosphate (Fig. 4 B), with a Ki of 2.8 ± 0.2 μM. Together with the product inhibition data, this pattern suggests that there is random substrate binding to the enzyme. If binding were ordered, desulfo-CoA would be an noncompetitive inhibitor versus one of the substrates. If the reaction proceeded via a ping-pong mechanism, noncompetitive inhibition would be also expected for the analogue versus one of the substrates.
In summary, in this report we describe the first kinetic analysis of a Pta from a member of the domain Archaea, and the data support the hypothesis that there is a kinetic mechanism that proceeds through random addition of both substrates to the enzyme prior to any chemical step (Fig. 5). Furthermore, the oligomeric state of the enzyme has been clarified. The results of this study are essential for interpretation of the recently solved crystal structures of M. thermophila Pta in complex with the substrate CoA in order to elucidate the catalytic mechanism (8).
ACKNOWLEDGMENTS
This work was funded by NIH grants GM44661-09 to J.G.F.
REFERENCES
Cleland, W. W. 1977. Determining the chemical mechanisms of enzyme-catalyzed reactions. Adv. Enzymol. Relat. Areas Mol. Biol. 45:277-387.
Henkin, J., and R. H. Abeles. 1976. Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase. Biochemistry 15:3472-3479.
Iyer, P. P., and J. G. Ferry. 2001. Role of arginines in coenzyme A binding and catalysis by the phosphotransacetylase from Methanosarcina thermophila. J. Bacteriol. 183:4244-4250.
Iyer, P. P., S. H. Lawrence, K. B. Luther, K. R. Rajashankar, H. P. Yennawar, J. G. Ferry, and H. Schindelin. 2004. Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila. Structure 12:559-567.
Iyer, P. P., S. H. Lawrence, H. P. Yennawar, and J. G. Ferry. 2003. Expression, purification, crystallization and preliminary X-ray analysis of phosphotransacetylase from Methanosarcina thermophila. Acta Crystallogr. D Biol. Crystallogr. 59:1517-1520.
Kyrtopoulos, S. A., and D. P. Satchell. 1972. Kinetic studies with phosphotransacetylase. IV. Inhibition by products. Biochim. Biophys. Acta 276:383-391.
Latimer, M. T., and J. G. Ferry. 1993. Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila. J. Bacteriol. 175:6822-6829.
Lawrence, S. H., K. B. Luther, H. Schindelin, and J. G. Ferry. 2006. Structural and functional studies sugest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophlia. J. Bacteriol. 188:1143-1154.
Leatherbarrow, R. J. 2002. GraFit, 5.0.4 ed. Erithacus Software, Horley-Surry, United Kingdom.
Pelroy, R. A., and H. R. Whiteley. 1972. Kinetic properties of phosphotransacetylase from Veillonella alcalescens. J. Bacteriol. 111:47-55.
Rasche, M. E., K. S. Smith, and J. G. Ferry. 1997. Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila. J. Bacteriol. 179:7712-7717.
Segel, I. H. 1975. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. John Wiley & Sons, Inc., Toronto, Canada.
Stadtman, E. R. 1952. The purification and properties of phosphotransacetylase. J. Biol. Chem. 196:527-534.
Xu, Q. S., D. H. Shin, R. Pufan, H. Yokota, R. Kim, and S. H. Kim. 2004. Crystal structure of a phosphotransacetylase from Streptococcus pyogenes. Proteins 55:479-481.(Sarah H. Lawrence and Jam)
ABSTRACT
Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. A steady-state kinetic analysis of the phosphotransacetylase from Methanosarcina thermophila indicated that there is a ternary complex kinetic mechanism rather than a ping-pong kinetic mechanism. Additionally, inhibition patterns of products and a nonreactive substrate analog suggested that the substrates bind to the enzyme in a random order. Dynamic light scattering revealed that the enzyme is dimeric in solution.
TEXT
Together with acetate kinase (equation 2), phosphotransacetylase (Pta) plays an essential role (equation 1) in the conversion of acetyl coenzyme A (acetyl-CoA) to acetate and in the synthesis of ATP in fermentative anaerobes belonging to the domain Bacteria. Acetate kinase and Pta also activate acetate to acetyl-CoA (reverse of equations 1 and 2) for conversion to methane and carbon dioxide in the energy-yielding metabolism of Methanosarcina species belonging to the domain Archaea.
(1)
(2)
Pta was first purified from a fermentative organism, Clostridium kluyveri, in the 1950s (13). Early kinetic analyses of the Ptas from C. kluyveri and Veillonella alcalescens belonging to the domain Bacteria are consistent with the presence of a ternary complex kinetic mechanism (2, 6, 10). Mechanistic analyses of the enzyme were abandoned until cloning and heterologous expression of Pta from Methanosarcina thermophila, a methane-producing organism belonging to the domain Archaea, which allowed application of modern biochemical techniques (7). The recently published crystal structure of M. thermophila Pta, along with kinetic analyses of site-specific replacement variants (3, 8, 11), have made this enzyme the preferred model for elucidation of the catalytic mechanism of Pta. As Ptas from diverse fermentative microbes belonging to the domain Bacteria exhibit high levels of sequence identity (4), an understanding of the M. thermophila Pta can be extrapolated to all Ptas. The steady-state kinetic analysis of M. thermophila Pta presented here suggests that the kinetic mechanism proceeds through random formation of a ternary complex. Our results provide a kinetic foundation essential for interpreting structural information for the M. thermophila Pta (4, 8) in order to elucidate the catalytic mechanism.
The Pta from M. thermophila was heterologously expressed and purified as described previously (5), and the preparation appeared to be homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The homogeneity and approximate hydrodynamic radius of Pta were examined by dynamic light scattering (DLS) using a DynaPro-MS800 molecular sizing instrument (Protein Solutions, Lakewood, NJ) as follows. A 40-μl aliquot of Pta (2.5 mg/ml) in 25 mM Tris-HCl (pH 7.2) containing 180 mM KCl was centrifuged (10,000 x g, 10 min), and an aliquot was loaded into a 12-μl quartz cuvette. The hydrodynamic radius, molecular weight, and size distribution were determined by the means of at least 10 DLS measurements. Data analysis was performed using Dynamics 5.0 (Protein Solutions, Lakewood, NJ). A sample DLS data set is shown in Fig. 1. Although the enzyme was initially reported to exist in solution as a monomer (7), Pta was found to have a hydrodynamic radius of 3.7 ± 0.1 nm, corresponding to a molecular mass of 71 ± 3 kDa, which is twice the calculated molecular mass of a Pta monomer. The observed molecular mass indicated that Pta exists in solution as a dimer, which is consistent with the dimeric states observed for the crystal structures of Ptas from M. thermophila and Streptococcus pyogenes (4, 14).
The rates for both the forward (acetyl-CoA-forming) and reverse (acetyl phosphate-forming) directions of the reaction catalyzed by Pta were measured at 25°C by monitoring the change in absorbance at 233 nm concomitant with formation or hydrolysis of the thioester bond of acetyl-CoA ( = 4,360 M–1), using a 0.1-cm-path-length quartz cuvette in a Hewlett-Packard 8452A diode array spectrophotometer. The standard reaction mixture (200 μl) contained 50 mM Tris-HCl (pH 7.2), 20 mM NH4Cl, 20 mM KCl, 2 mM dithiothreitol, the appropriate substrate for the experiment, and a concentration of Pta sufficient to yield a linear rate over at least 2 min (usually 0.05 μg/ml). Reactions were initiated by addition of the second substrate. All components were maintained on ice and warmed to 25°C immediately prior to initiation of the reaction.
The initial velocity patterns of the two-substrate, two-product reaction catalyzed by Pta were investigated in order to differentiate between a ternary complex kinetic mechanism and a ping-pong kinetic mechanism. For each direction of the reaction catalyzed by Pta, the initial velocity of the reaction was measured by using a matrix of five different concentrations of each substrate. Rates were measured using the standard activity assay, and each reaction was initiated by addition of the varied substrate. Data were expressed as double-reciprocal plots and fitted globally using Grafit 5.0 (9) to equation 3 describing the pattern for a ternary complex kinetic mechanism (1):
(3)
where V is the maximal velocity, A and B are the concentrations of the varied and fixed substrates, respectively, KA and KB are the Michaelis constants for substrates A and B, respectively, and KD(A) is the dissociation constant for the varied substrate.
For both directions, the data yielded sets of intersecting lines fitted to equation 1 (Fig. 2). For the forward direction, Michaelis constants of 186 ± 6 and 65 ± 7 μM were obtained for acetyl phosphate and CoA, respectively, and the kcat was 5,190 ± 30 s–1. For the reverse direction, Michaelis constants of 96 ± 13 and 742 ± 86 μM were obtained for acetyl-CoA and phosphate, respectively, and the kcat was 1,500 ± 30 s–1. The initial velocity patterns were similar to those observed for the Ptas from V. alcalescens and C. kluyveri (2, 10) and are consistent with a kinetic mechanism that proceeds via formation of a ternary complex between Pta and both substrates prior to any chemical step, rather than via a ping-pong mechanism.
The product inhibition patterns of the reaction catalyzed by Pta were analyzed to determine if substrate binding and product release are random or ordered. Inhibition of the forward (acetyl-CoA-forming) reaction catalyzed by Pta by the product inhibitors, acetyl-CoA and inorganic phosphate, was analyzed with respect to various concentrations of the substrates, CoA and acetyl phosphate, using the standard activity assay. All four product-substrate pairs were analyzed at saturating and subsaturating conditions using a matrix of five concentrations of substrates and inhibitors for each experiment. Data were expressed as double-reciprocal plots and analyzed to determine the nature of the inhibition. Data were fitted using Grafit 5.0 (9) to equations describing competitive (equation 4) or noncompetitive (equation 5) inhibition using two-dimensional least-squares analysis (12).
(4)
(5)
where Km is the Michaelis constant for the substrate, S is the concentration of the substrate, I is the concentration of the inhibitor, and Ki is the inhibition constant for the product inhibitor.
Phosphate was a competitive inhibitor versus acetyl phosphate when CoA was at saturating (600 μM) or subsaturating (60 μM) levels (Fig. 3A and B). Phosphate was a noncompetitive inhibitor versus CoA when acetyl phosphate was at a subsaturating level (150 μM) (Fig. 3C), but it did not inhibit versus CoA when acetyl phosphate was at a saturating level (4mM). Acetyl-CoA was a competitive inhibitor versus CoA when acetyl phosphate was at subsaturating levels (Fig. 3D), but it did not inhibit versus CoA when acetyl phosphate was at saturating levels. Similarly, acetyl-CoA was a competitive inhibitor versus acetyl phosphate when CoA was at subsaturating levels (Fig. 3E), but it did not inhibit versus acetyl phosphate when CoA was at saturating levels. This pattern of inhibition is diagnostic for a kinetic mechanism that proceeds through formation of a ternary complex in which the substrates can bind to the enzyme in random order (1, 12).
The inhibition patterns of the nonreactive CoA analogue desulfo-CoA were analyzed with respect to CoA and acetyl phosphate, and the results further supported the random binding suggested by the product inhibition patterns. Data were expressed as double-reciprocal plots and fitted to equations 4 and 5 describing competitive and noncompetitive inhibition, respectively. Desulfo-CoA was a competitive inhibitor with respect to CoA (Fig. 4A), with a Ki of 1.3 ± 0.1 μM, confirming a previous report (3). Desulfo-CoA was a noncompetitive inhibitor with respect to acetyl phosphate (Fig. 4 B), with a Ki of 2.8 ± 0.2 μM. Together with the product inhibition data, this pattern suggests that there is random substrate binding to the enzyme. If binding were ordered, desulfo-CoA would be an noncompetitive inhibitor versus one of the substrates. If the reaction proceeded via a ping-pong mechanism, noncompetitive inhibition would be also expected for the analogue versus one of the substrates.
In summary, in this report we describe the first kinetic analysis of a Pta from a member of the domain Archaea, and the data support the hypothesis that there is a kinetic mechanism that proceeds through random addition of both substrates to the enzyme prior to any chemical step (Fig. 5). Furthermore, the oligomeric state of the enzyme has been clarified. The results of this study are essential for interpretation of the recently solved crystal structures of M. thermophila Pta in complex with the substrate CoA in order to elucidate the catalytic mechanism (8).
ACKNOWLEDGMENTS
This work was funded by NIH grants GM44661-09 to J.G.F.
REFERENCES
Cleland, W. W. 1977. Determining the chemical mechanisms of enzyme-catalyzed reactions. Adv. Enzymol. Relat. Areas Mol. Biol. 45:277-387.
Henkin, J., and R. H. Abeles. 1976. Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase. Biochemistry 15:3472-3479.
Iyer, P. P., and J. G. Ferry. 2001. Role of arginines in coenzyme A binding and catalysis by the phosphotransacetylase from Methanosarcina thermophila. J. Bacteriol. 183:4244-4250.
Iyer, P. P., S. H. Lawrence, K. B. Luther, K. R. Rajashankar, H. P. Yennawar, J. G. Ferry, and H. Schindelin. 2004. Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila. Structure 12:559-567.
Iyer, P. P., S. H. Lawrence, H. P. Yennawar, and J. G. Ferry. 2003. Expression, purification, crystallization and preliminary X-ray analysis of phosphotransacetylase from Methanosarcina thermophila. Acta Crystallogr. D Biol. Crystallogr. 59:1517-1520.
Kyrtopoulos, S. A., and D. P. Satchell. 1972. Kinetic studies with phosphotransacetylase. IV. Inhibition by products. Biochim. Biophys. Acta 276:383-391.
Latimer, M. T., and J. G. Ferry. 1993. Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila. J. Bacteriol. 175:6822-6829.
Lawrence, S. H., K. B. Luther, H. Schindelin, and J. G. Ferry. 2006. Structural and functional studies sugest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophlia. J. Bacteriol. 188:1143-1154.
Leatherbarrow, R. J. 2002. GraFit, 5.0.4 ed. Erithacus Software, Horley-Surry, United Kingdom.
Pelroy, R. A., and H. R. Whiteley. 1972. Kinetic properties of phosphotransacetylase from Veillonella alcalescens. J. Bacteriol. 111:47-55.
Rasche, M. E., K. S. Smith, and J. G. Ferry. 1997. Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila. J. Bacteriol. 179:7712-7717.
Segel, I. H. 1975. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. John Wiley & Sons, Inc., Toronto, Canada.
Stadtman, E. R. 1952. The purification and properties of phosphotransacetylase. J. Biol. Chem. 196:527-534.
Xu, Q. S., D. H. Shin, R. Pufan, H. Yokota, R. Kim, and S. H. Kim. 2004. Crystal structure of a phosphotransacetylase from Streptococcus pyogenes. Proteins 55:479-481.(Sarah H. Lawrence and Jam)