Research Article: Detailed Enzyme Kinetics in Terms of Biochemical Species: Study of Citrate Synthase

Date Published: March 19, 2008

Publisher: Public Library of Science

Author(s): Daniel A. Beard, Kalyan C. Vinnakota, Fan Wu, Timothy Secomb.

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.

Partial Text: While the study of the catalytic kinetics of enzymes represents one of the most established and well documented fields in biochemical research, the impact of biochemical state (pH, ionic strength, temperature, and certain cation concentrations) is typically not formally accounted for in kinetic studies [1], [2]. In vitro experiments using purified proteins and controlled substrate concentrations to characterize enzyme kinetics are conducted under conditions that do not necessarily match the physiological environment, but are determined based on a number of factors, including the requirements of the assays used to measure the kinetics. Therefore it is difficult to compare results obtained from different studies and to use available kinetic data to predict in vivo function without ambiguity.

Here we have introduced a model for the compulsory order ternary-complex catalytic mechanism that is formulated in terms of chemical species, allowing the model to account for variable state, including pH and metal ion concentrations. In addition, a general form of the model, with potential inhibitory binding at each enzyme state is introduced. The model is used to analyze independent data sets from a number of labs on different isoforms of citrate synthase and develop a consensus mechanism that explains the available data. This consensus mechanism provides a detailed understanding of the basic mechanism of this enzyme and can be useful in computational simulation of biochemical systems including this enzyme.