BC351 Lecture 10 Metabolism Metabolic Control Terms Allosteric enzyme Steady State Flux Energy Charge Principles 1 Enzymatic regulation at the molecular level a Allosteric regulation and covalent modifications 2 Steady state vs flux of metabolic pathways a Which way will metabolites flow b How this relates to enzymatic regulation c How ATP AMP are major allosteric effectors or modulators 3 Our specific example PFK 1 FBPase 1 a Insulin vs Glucagon signaling I Introduction pgs 569 570 a The need for control i Glucose fates in the liver cell 1 How does the cell decide which path should be taken LN10 1 ii PFK 1 FBPase 1 bypass reactions PFK 1 G 8 6 kJ mol G 22 kJ mol F6P FBPase 1 F16BP ATP ADP F16BP F6P Pi ATP ADP Pi 1 Both reactions are irreversible in opposite direction 2 The reactant of one is the product of the other 3 IF both were running in the same cell at the same time a futile cycle would be made a Clearly this would be pointless and needs to be avoided LN10 2 b An illustration of control i The Pasteur effect No O2 anaerobic O2 1 Louis Pasteur was able to show that a yeast culture will consume 10x more glucose when in an anaerobic environment as compared to an aerobic environment a Clearly this is a dramatic change in metabolic output b Organisms of all kinds demonstrate the capacity to undergo dramatic changes in their metabolic output in response to changes in their external environment but how do they do this i Enzyme regulation LN10 3 II Enzymatic regulation pgs 220 224 571 574 a Five paths of regulation i Protein turnover the number of enzymes available 1 Rate of synthesis vs rate of degredation ii Protein sequestration effective activity within a subcellular compartment 1 Enzymes can be sequestered to a cellular location that does not have appreciable concentrations of its substrate iii Protein interaction with a regulatory protein iv By allosteric regulation 1 Allosteric enzyme definition a LN10 4 2 Allosteric enzymes activity can be increased or decreased by the binding of modulators a Modulators can be i Negative inhibitory ii Positive activating 3 Allosteric enzyme kinetics a Generally vo vs S graphs are sigmoidal i Usually these proteins are cooperative and small changes in S translate to large changes in vo 1 In other words they can be very sensitive to environmental changes b These enzymes kinetics cannot be described by the Michaelis Menten equation however i Vmax can still be obtained 1 Vmax kcat E T ii Km is now referred to as K0 5 1 It still represents the S required to reach Vmax c Modulation leads to i Increases decreases in Vmax ii Increases decreases in K0 5 iii Or a combination LN10 5 v By covalent modifications 1 Over 500 different types of covalent modifications a Phosphorylation Adenylylation Acetylation ADPribosylation Methylation b These modifications can have an inhibitory or activating affect on the enzyme 2 Phosphorylation is by far the most common a of all known proteins are phosphorylated b Kinases and phosphotases i These enzymes allow for the reversible phosphorylation dephosphorylation of regulatory enzymes LN10 6 III Principles of metabolic regulation pgs 502 503 571 574 576 a Control is complicated i Regulatory genes in humans 1 In excess of 4000 2 At least 500 different kinases b Steady state and metabolic flux i Metabolic pathways and indeed biological organisms are kept at a steady state 1 Definition of a Steady state a 2 Substrate concentrations are maintained a The fixed concentration determines the Q for each biological reaction Glucose G6P F6P F16BP GAP DHAP Fixed concentration b Q for some reactions ensure irreversibility meaning that the driving force for that reaction is set toward CONCLUSION The potential of metabolic intermediates to flow in a certain direction does not change product production c The driving force for these irreversible reactions determines the thermodynamically preferred pathway direction due to the reversibility of the other reactions d An implication of this is that enzymes that catalyze irreversible reactions are usually regulated in some way i All other enzymes are regulated indirectly by the direct regulation of the irreversible enzymes LN10 7 ii Metabolic flux 1 Definition of flux a iii Flux is directly related to enzymatic regulation 1 An example Ribose 5 phosphate Glucose G6P F6P F16BP GAP DHAP Glycogen a Glycolysis PPP or glycogen synthesis b Irreversible enzymes are gates in a complicated canal system IV ATP ADP AMP as central control effectors pgs 502 503 571 574 577 935 a What is the unifying principle of all metabolic anabolic catabolic pathways i Utilization and conservation of free energy 1 When the cell has lots of free energy potential to do work it would be expected that anabolic pathways would be upregulated 2 When the cell has small amounts of free energy potential to do work it would be expected that catabolic pathways would be upregulated LN10 8 ii Energy charge ATP and anabolic catabolic pathway utilization 1 Definition of energy charge a 2 This is a reflection of the energy status within the cell of Total 100 50 0 0 0 5 ENERGY CHARGE 1 0 3 Notice that WHEN a ATP is high the cell is considered to have a high energy charge values closer to 1 0 b AMP are high the cell is considered to have a low energy charge values closer to 0 0 LN10 9 iii Cellular energy charge 1 Typical values of energy charge are between 0 85 and 0 95 2 Between these two values we see a fairly large change in metabolic output so that when energy charge is a High 0 95 the ATP utilizing pathways anabolic will be upregulated as we said before i The irreversible enzymes in these paths will be activated ii These enzyme often times will have either ATP as a positive modulator or AMP ADP as negative modulators b Low 0 85 the ATP generating pathways catabolic will be upregulated as we said before i The irreversible enzymes in these paths will be activated ii These enzyme often times will have either ATP as a negative modulator or AMP ADP as positive modulators 3 Conclusion ATP a ATP and ADP AMP are strong allosteric effectors ANABOLIC CATABOLIC ANABOLIC regulate catabolic anabolic pathway utilization and prevents dangerous drops in ATP ADP AMP of irreversible enzymes This allows the cell to CATABOLIC LN10 10 b OTHER THAN ITS AFFECT ON METABOLIC CONTROL WHY IS ATP CONCENTRATION SO CRITICAL TO THE LIFE OF THE CELL i For two different reasons 1 Kinetic GLUCOSE ATP G6P ADP Free energy G 2 Thermodynamic Glucose ATP G6P ADP LN10