A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell

Abstract

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course.