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Detailed enzyme kinetics in terms of biochemical species: study of citrate synthase.

Beard DA, Vinnakota KC, Wu F - PLoS ONE (2008)

Bottom Line: The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant.The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data.This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology and Bioengineering Center and Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America. dbeard@mcw.edu

ABSTRACT
The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant. The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data. This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme.

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Related in: MedlinePlus

Analysis using random-order model of Equation (26).Data and conditions in A, B, C, and D are the same as for Figure 2. Data and conditions for E and F are the same as for Figures 3A and 3B, respectively Parameter values for solid line model predictions are Vmax = 0.320 µmol·min−1· µg−1, KmB = 6.20 µM, KmP = 8.00 µM, KeA = 1.35 µM, KeB = 1.10 µM, KeP = 21.6 nM, KeQ = 0.150 µM. Parameter values for dashed line model predictions are Vmax = 0.526 µmol·min−1·µg−1, KmB = 36.6 µM, KmP = 80.792 mM, KeA = 3.08 nM, KeB = 10.8 nM, KeP = 0.152 µM, KeQ = 17.0 µM.
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pone-0001825-g007: Analysis using random-order model of Equation (26).Data and conditions in A, B, C, and D are the same as for Figure 2. Data and conditions for E and F are the same as for Figures 3A and 3B, respectively Parameter values for solid line model predictions are Vmax = 0.320 µmol·min−1· µg−1, KmB = 6.20 µM, KmP = 8.00 µM, KeA = 1.35 µM, KeB = 1.10 µM, KeP = 21.6 nM, KeQ = 0.150 µM. Parameter values for dashed line model predictions are Vmax = 0.526 µmol·min−1·µg−1, KmB = 36.6 µM, KmP = 80.792 mM, KeA = 3.08 nM, KeB = 10.8 nM, KeP = 0.152 µM, KeQ = 17.0 µM.

Mentions: Measured flux as a function of substrate concentrations was obtained from Figures 2, 3, 6, 7, and 9 of [14]. Initial fluxes (µmol of COASH (or CIT) synthesized per minute per µg of enzyme) measured at the substrate concentrations indicated in the figures. For A, B, and D, the initial product (CIT and COASH) concentrations are zero. C. Flux measured with COASH added in various concentrations to investigate the kinetics of product inhibition. All data were obtained at pH = 8.1 at 28°C. Model fits are plotted as solid lines.


Detailed enzyme kinetics in terms of biochemical species: study of citrate synthase.

Beard DA, Vinnakota KC, Wu F - PLoS ONE (2008)

Analysis using random-order model of Equation (26).Data and conditions in A, B, C, and D are the same as for Figure 2. Data and conditions for E and F are the same as for Figures 3A and 3B, respectively Parameter values for solid line model predictions are Vmax = 0.320 µmol·min−1· µg−1, KmB = 6.20 µM, KmP = 8.00 µM, KeA = 1.35 µM, KeB = 1.10 µM, KeP = 21.6 nM, KeQ = 0.150 µM. Parameter values for dashed line model predictions are Vmax = 0.526 µmol·min−1·µg−1, KmB = 36.6 µM, KmP = 80.792 mM, KeA = 3.08 nM, KeB = 10.8 nM, KeP = 0.152 µM, KeQ = 17.0 µM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266798&req=5

pone-0001825-g007: Analysis using random-order model of Equation (26).Data and conditions in A, B, C, and D are the same as for Figure 2. Data and conditions for E and F are the same as for Figures 3A and 3B, respectively Parameter values for solid line model predictions are Vmax = 0.320 µmol·min−1· µg−1, KmB = 6.20 µM, KmP = 8.00 µM, KeA = 1.35 µM, KeB = 1.10 µM, KeP = 21.6 nM, KeQ = 0.150 µM. Parameter values for dashed line model predictions are Vmax = 0.526 µmol·min−1·µg−1, KmB = 36.6 µM, KmP = 80.792 mM, KeA = 3.08 nM, KeB = 10.8 nM, KeP = 0.152 µM, KeQ = 17.0 µM.
Mentions: Measured flux as a function of substrate concentrations was obtained from Figures 2, 3, 6, 7, and 9 of [14]. Initial fluxes (µmol of COASH (or CIT) synthesized per minute per µg of enzyme) measured at the substrate concentrations indicated in the figures. For A, B, and D, the initial product (CIT and COASH) concentrations are zero. C. Flux measured with COASH added in various concentrations to investigate the kinetics of product inhibition. All data were obtained at pH = 8.1 at 28°C. Model fits are plotted as solid lines.

Bottom Line: The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant.The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data.This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology and Bioengineering Center and Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America. dbeard@mcw.edu

ABSTRACT
The compulsory-ordered ternary catalytic mechanism for two-substrate two-product enzymes is analyzed to account for binding of inhibitors to each of the four enzyme states and to maintain the relationship between the kinetic constants and the reaction equilibrium constant. The developed quasi-steady flux expression is applied to the analysis of data from citrate synthase to determine and parameterize a kinetic scheme in terms of biochemical species, in which the effects of pH, ionic strength, and cation binding to biochemical species are explicitly accounted for in the analysis of the data. This analysis provides a mechanistic model that is consistent with the data that have been used support competing hypotheses regarding the catalytic mechanism of this enzyme.

Show MeSH
Related in: MedlinePlus