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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.

Show MeSH
Basic compulsory-order ternary-complex mechanism.The basic ordered mechanism for the general reaction , with a = [A], b = [B], p = [P], and q = [Q] is illustrated. The four states refer to unbound enzyme (state 1), enzyme-substrate A complex (E·A, state 2), enzyme-substrate A-substrate B complex (E·AB, state 3), and enzyme-product Q complex (E·Q, state 4). The four steps of the catalytic cycle are detailed in Equation (1).
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pone-0001825-g001: Basic compulsory-order ternary-complex mechanism.The basic ordered mechanism for the general reaction , with a = [A], b = [B], p = [P], and q = [Q] is illustrated. The four states refer to unbound enzyme (state 1), enzyme-substrate A complex (E·A, state 2), enzyme-substrate A-substrate B complex (E·AB, state 3), and enzyme-product Q complex (E·Q, state 4). The four steps of the catalytic cycle are detailed in Equation (1).

Mentions: The basic compulsory-order ternary-complex mechanism, also called the ordered bi-bi mechanism, is illustrated in Figure 1 for the general reaction . The mechanism involves four enzyme state transitions:(1)where each state transition is assumed to proceed by mass action [10], [11]. Here E1 represents free (unbound) enzyme; E2 represents the complex formed between enzyme and the species A, which binds first; E3 is the ternary complex that represents enzyme bound to both substrates or both products; and E4 represents the complex formed between enzyme and the species Q. In Figure 1 the substrate and product concentrations are denoted a = [A], b = [B], p = [P], and q = [Q] and the reactant concentrations are incorporated into apparent mass-action rate constants for the state transitions between enzyme states 1, 2, 3, and 4.


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

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

Basic compulsory-order ternary-complex mechanism.The basic ordered mechanism for the general reaction , with a = [A], b = [B], p = [P], and q = [Q] is illustrated. The four states refer to unbound enzyme (state 1), enzyme-substrate A complex (E·A, state 2), enzyme-substrate A-substrate B complex (E·AB, state 3), and enzyme-product Q complex (E·Q, state 4). The four steps of the catalytic cycle are detailed in Equation (1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001825-g001: Basic compulsory-order ternary-complex mechanism.The basic ordered mechanism for the general reaction , with a = [A], b = [B], p = [P], and q = [Q] is illustrated. The four states refer to unbound enzyme (state 1), enzyme-substrate A complex (E·A, state 2), enzyme-substrate A-substrate B complex (E·AB, state 3), and enzyme-product Q complex (E·Q, state 4). The four steps of the catalytic cycle are detailed in Equation (1).
Mentions: The basic compulsory-order ternary-complex mechanism, also called the ordered bi-bi mechanism, is illustrated in Figure 1 for the general reaction . The mechanism involves four enzyme state transitions:(1)where each state transition is assumed to proceed by mass action [10], [11]. Here E1 represents free (unbound) enzyme; E2 represents the complex formed between enzyme and the species A, which binds first; E3 is the ternary complex that represents enzyme bound to both substrates or both products; and E4 represents the complex formed between enzyme and the species Q. In Figure 1 the substrate and product concentrations are denoted a = [A], b = [B], p = [P], and q = [Q] and the reactant concentrations are incorporated into apparent mass-action rate constants for the state transitions between enzyme states 1, 2, 3, and 4.

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