Lecture 8 in Powerpoint format

A b c m d x if the mar is close to keq then reaction

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Unformatted text preview: n, Calculate and compare to published K’eq values. and eq values. A B C M… D X… If the MAR is close to K’eq, then reaction is then close to equilibrium and unlikely to be a major control step. control If the MAR is 100-10,000 times lower than If KEq then the reaction is possibly ratecontrolling. MAR = [B] <<< Keq MAR [B] [A] [A] is relatively high, and/or [B] is relatively low A few more notes in this regard… few A reaction having a MAR that is much smaller than Keq, suggests reaction MAR suggests that the reaction is going as fast as it can, but that this is inadequate to approach equilibrium. Thus, any increased activity of this enzyme could result in a large Thus, increase in the MAR, which can potentially influence greatly the increase which other enzymes in the pathway overall. (since many are ‘responders’) ‘responders’) All reactions with a strong degree of control over pathway flux are All thus far from equilibrium, far Many of these enzymes require activation by regulatory molecules Many (next slide) (next s sR s s s s s s s R R R s Imagine an enzyme that is far from equilibrium, and is saturated Imagine with substrate. Thus, it is working at Vmax, and an increase in [S] does not affect Thus, its activity. (A great decrease in S would however cause great decreased activity) decreased The enzyme could be activated by regulatory molecules ( e.g., to increase Vmax) increase Thus, all reactions with a strong degree of control over pathway Thus, flux are far from equilibrium, BUT not all reactions which are far from equilibrium are rate controlling. (Many such enzymes require regulators) regulators) 5. Perturb the pathway to identify the ‘cross-over point’ ‘cross-over Stimulate (or inhibit) system so that flux is increased (or decreased) Stimulate E.g., epinephrine to study muscle glycolysis. E.g., The activity of controlling enzyme should increase, and thus increase The [products]/[substrates] [products]/[substrates] Metabolite concentrations As % of unstimulated concentrations A B C D E F “Cross-over Plot” 100 A B C D E F Rate controlling step Problems with traditional approaches for the identification of rate-controlling steps steps Many involve too much guesswork! – non-empirical Practicalities: it is very difficult to measure accurately the concentrations of all metabolites in a pathway metabolites Accuracy: Intracellular compartmentalization issues: there is sometimes contamination during the isolation of the desired intracellular compartment by other (irrelevant) compartments. An example An Application of these concepts Application to the control of metabolic flux through glycolysis in the heart glycolysis heart Enzyme Vmax Keq MAR HK 7 4000 0.08 Phosphoglucose isomerase 65 0.4 0.24 PFK 14 1000 0.03 Aldolase 24 0.0001 0.00001 Triose phosphate isomerase 580 0.04 0.24 G3PDH/ phosphoglycerate kinase 135/74 0.005 0.111 Phosphoglycerate mutase 27 0.2 0.12 Enolase 15 3.0 1.4 Pyruvate kinase 145 10 000 40...
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