Metabolic Control Analysis

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It is widely believed that the steps in the major metabolic pathways are known and that the control of flux through these pathways occurs at a very limited number of "rate limiting" steps. This concept has lead to the design of drugs to alter the kinetics of these "rate limiting enzymes". It has also led to attempts to alter metabolic pathways by altering the amounts of rate limiting enzymes using the techniques of molecular biology. To the dismay of many, such interventions often fail to alter the rates of the pathways under study. These failures have led to an increased awareness that "control" of pathway flux is distributed among many enzymes of a metabolic pathway and can vary from enzyme to enzyme depending upon conditions (Brand, M. Protein Science, 6: 1368-1369, 1997; Shulman, R.G. FASEB J. 258: 255-258, 1998). Metabolic control theory had long predicted distribution of control among many enzymes of a pathway (Veech, R.L. & Fell, D.A. Cell Biochem. & Function 14: 229-236, 1996). However, actual demonstration and testing of such theories was technically difficult. We were the first laboratory to make the required measurements of flux, kinetic and thermodynamic constants of each step, and the levels of all substrates and products required to make such a formal analysis of flux control in a major metabolic pathway (Kashiwaya, Y. et al, J. Biol. Chem. 269: 25502-25514, 1994). However, the detailed analysis of an entire metabolic pathway was labor intenstive and extremely time consuming. Therefore during FY97 we began developing new and more automated methods of metabolite analysis which will allow us to perform the measurements required for metabolic control analysis in a more rapid manner. We are also attempting to develop micro methods suitable for analysis of cells in culture, and specifically neuronal cells. We will then apply these new methods to determine if the provision of acetyl units to neural tissue can alter the course of a variety of neurological conditions including alcohol withdrawal syndrome.