Lecture 8 in Powerpoint format

try perturbing flux and determining which

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Unformatted text preview: ?? Try perturbing flux and determining which metabolites increase and which decrease (i.e., construct a cross-over plot) In a perfused heart system (i.e., in vitro): Add insulin to the perfusate to support glucose uptake Measure baseline concentrations of metabolites Then, induce anoxia to perturb (increase) flux through glycolysis, measure concentrations of metabolites and construct cross-over plot • ADP and fructose 1,6 bisphosphate increase • ATP and fructose 6-P concentrations fall HK Continued… ADP and fructose 1,6 bisphosphate increase ATP and fructose 6-P concentrations fall PFK HK and PFK are identified as the two major rate controlling steps. Pyruvate kinase is not a major rate controlling step under these conditions since PEP did not fall and ADP increased (not decreased). Metabolic control analysis Metabolic For too long the central idea in discussions of metabolic regulation has For been that everything can be understood in terms of the properties of a few components. As Henrik Kacser remarked, “One thing is certain: to understand the whole, you must look at the whole.” whole, Origins: Kacser and Burns; Newsholme; Kell; Brand Common points with Traditional Approaches: Reaction thermodynamics are important Major rate controlling enzymes are far from equilibrium What is metabolic control analysis? What A method for analyzing the proportional distribution of control method over flux (and metabolite concentrations) in a pathway. Instead of assuming the existence of one rate-limiting step, it Instead assumes that control is spread amongst the various steps. assumes Metabolic control analysis was formerly (and is sometimes still) known as metabolic control theory. It is closely related to the metabolic It engineering discipline known as sensitivity analysis. sensitivity Two types : ‘Bottom-up’ and ‘Top-down’ ‘Bottom-up’ ‘Top-down’ The former involves assessing the control strength of every The individual enzymatic step, and requires the use of very specific individual and very inhibitors for each and every enzyme inhibitors ? A ? B ? C ? E D The latter looks at the pathway as a simplified system with a The as reactant and a product with one (or two) intermediate metabolite(s). It breaks up the pathway into two or three reaction blocks, and It determine the importance of each block (e.g. 80% & 20%). You can continue as needed to break up the blocks into smaller and You smaller block of reactions, to provide more detailed assessments of the distribution of control. ? A ? C E Advantages and Disadvantages Disadvantages Jc JL Bottom-up: A lot of tedious work! Often there are not highly specific inhibitors for all steps, making this approach impossible. Top-down: Allows you to quickly and easily identify the most important sites of control Do not need many inhibitors Can more easily be applied to complex systems What are the main questions that What metabolic control analysis aims to answer? (Both bottom-up and top-down will (Both ultimately provide the same answers) answers) Metabolic control analysis begins by recognizing that flux control is not a unique property of one rate-limiting enzyme, but is distributed among all of the enzymes. It then set...
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