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1、Principles of Metabolic Regulation chapter 15 S 173 1 Measurement of Intracellular Metabolite Concentrations Measuring the concentrations of metabolic intermediates in a living cell presents great experimental difficulties usually a cell must be destroyed before metabolite concentrations can be meas
2、ured Yet enzymes catalyze metabolic interconversions very rapidly so a common problem associated with these types of measurements is that the findings reflect not the physiological concentrations of metabolites but the equilibrium con centrations A reliable experimental technique requires all enzyme
3、 catalyzed reactions to be instanta neously stopped in the intact tissue so that the metabolic intermediates do not undergo change This objective is accomplished by rapidly compressing the tissue between large aluminum plates cooled with liquid nitrogen 190 C a process called freeze clamping After f
4、reezing which stops enzyme action instantly the tissue is powdered and the enzymes are inactivated by precipitation with perchlo ric acid The precipitate is removed by centrifugation and the clear supernatant extract is analyzed for metabolites To calculate intracellular concentrations the intracell
5、ular volume is determined from the total water content of the tissue and a measurement of the extracellular volume The intracellular concentrations of the substrates and products of the phosphofructokinase 1 reac tion in isolated rat heart tissue are given in the table below MetaboliteConcentration
6、mM Fructose 6 phosphate87 0 Fructose 1 6 bisphosphate22 0 ATP11 400 ADP1 320 Source From Williamson J R 1965 Glycolytic control mechanisms I inhibition of glycolysis by acetate and pyruvate in the isolated perfused rat heart J Biol Chem 240 2308 2321 Calculated as mmol mL of intracellular water a Ca
7、lculate Q fructose 1 6 bisphosphate ADP fructose 6 phosphate ATP for the PFK 1 reaction under physiological conditions b Given a G for the PFK 1 reaction of 14 2 kJ mol calculate the equilibrium constant for this reaction c Compare the values of Q and K eq Is the physiological reaction near or far f
8、rom equilibrium Explain What does this experiment suggest about the role of PFK 1 as a regulatory enzyme Answer a The mass action ratio Q 0 0293 22 0 mM 1 320 mM 87 0 mM 11 400 mM 2608T ch15sm S173 S182 02 22 2008 1 24 pm Page S 173 pinnacle 111 WHQY028 Solutions Manual Ch 15 S 174Chapter 15Principl
9、es of Metabolic Regulation b G RT ln K eq RT at 25 C 2 48 kJ mol ln K eq G RT 14 2 kJ mol 2 48 kJ mol 5 73 K eq e 5 73 308 c Because K eqis much greater than Q it is clear that the PFK 1 reaction does not approach equilibrium in vivo and thus the product of the reaction fructose 1 6 bisphosphate doe
10、s not approximate an equilibrium concentration Metabolic pathways are open systems operating under near steady state conditions with substrates flowing in and products flowing out at all times Thus all the fructose 1 6 bisphosphate formed is rapidly used or turned over The PFK 1 catalyzed reaction w
11、hich is the rate limiting step in glycolysis is an excellent candidate for the critical regulatory point of the pathway 2 Are All Metabolic Reactions at Equilibrium a Phosphoenolpyruvate PEP is one of the two phosphoryl group donors in the synthesis of ATP during glycolysis In human erythrocytes the
12、 steady state concentration of ATP is 2 24 mM that of ADP is 0 25 mM and that of pyruvate is 0 051 mM Calculate the concentration of PEP at 25 C assuming that the pyruvate kinase reaction see Fig 13 13 is at equilibrium in the cell b The physiological concentration of PEP in human erythrocytes is 0
13、023 mM Compare this with the value obtained in a Explain the significance of this difference Answer a First we must calculate the overall G for the pyruvate kinase reaction by breaking this process into two reactions and summing the G values from Table 13 6 1 PEP H2O 88npyruvate Pi G 1 61 9 kJ mol 2
14、 ADP Pi88nATP H2O G 2 30 5 kJ mol Sum PEP ADP 88npyruvate ATP G sum 31 4 kJ mol Assuming the reaction is at equilibrium we can calculate K eq G RT ln K eq ln K eq G RT 31 4 kJ mol 2 48 kJ mol 12 7 K eq 3 28 105 Because K eq where all reactants and products are at their equilibrium concentrations PEP
15、 1 4 10 9M b The physiological PEP of 0 023 mMis 16 000 times the equilibrium concentration This reaction like many others in the cell is not at equilibrium 0 023 10 3M 1 4 10 9M 5 1 10 5M 2 24 10 3M 2 5 10 4M 3 28 105 pyruvate ATP ADP K eq pyruvate ATP ADP PEP 2608T ch15sm S173 S182 02 22 2008 1 24
16、 pm Page S 174 pinnacle 111 WHQY028 Solutions Manual Ch 15 3 Effect of O2Supply on Glycolytic Rates The regulated steps of glycolysis in intact cells can be identified by studying the catabolism of glucose in whole tissues or organs For example the glucose consumption by heart muscle can be measured by artificially circulating blood through an isolated intact heart and measuring the concentration of glucose before and after the blood passes through the heart If the circulating blood is deoxygena
