lecture 16.105 Dark reactions, Regulation

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Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 1 Course evaluation forms. Please fill them out. Dark Reactions: These reactions were worked out as one of the earliest uses of radioactivity in biology/biochemistry. Calvin at Berkeley. Pulse algae with 14 CO 2 for a short time, and then dump into boiling alcohol and separate compounds. Look for radioactivity. Pulse 5s, 14C found in 3-PG. Pulse 30s, 14C found in 3-PG, triose phosphates (G-3-P, DHAP) and hexose phosphates (F-6-P, G-6-P) What happens from there? We haven’t talked about gluconeogenesis yet, but going up we see this as a lead in to making glucose from other reduced compounds and CO 2 . The compound to which CO 2 is attached is the 5-carbon D-ribulose-1,5- bisphosphate.

Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 2 The enzyme that does this is ribulose-1,5-bisphosphate carboxylase/oxygenase, a.k.a. Rubisco. Rubisco is the most abundant protein on Earth. Rubisco catalyzes the attachment of CO 2 to rbp, making an unstable 6-carbon intermediate, which cleaves to 2 molecules of 3-PG. What is happening here? We are attaching CO 2 one molecule at a time, and making a six carbon sugar. So we need six turns of the cycle to make one six carbon sugar. The entire scheme looks like this (and it’s been drawn as going six times) :

Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 3 So for every 6 CO 2 fixed: Consume 18 ATP Consume 12 NADPH Gain 1 6-Carbon Sugar (F-6-P) Regenerate 6 RuBP Oxygenation activity of RubisCO RubisCO accepts O 2 as a substrate in addition to CO 2 . When this happens, no carbon is fixed and it represents a loss of energy. Re-capture of the carbon from glycolate is expensive. The glycolate pathway involves carbon transport through peroxisomes to the mitochondria and back, and consumes ATP and produces CO 2 . Hence the name photorespiration. The Km of RubisCO for CO 2 is ~9  m, for O 2 ~350  M. The modern atmosphere contains 20% O 2 and 0.04% CO 2 , giving concentrations in aqueous solutions in equilibrium with air of ~250  m O 2 and ~11  m CO 2 . This means RubisCO reacts with CO 2 and O 2 in a ratio of about 3:1. Large loss for the planet. Certain types of plants have evolved metabolic pathways and other mechanisms to deal with this. Take plant physiology to learn more. This also plays into the discussion of the rise in atmospheric CO 2.

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Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 4 Metabolic Integration Three interconnected functional blocks; CATABOLISM, ANABOLISM & MACROMOLECULAR SYNTHESIS

Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 5 CATABOLISM – Food oxidized to CO 2 and H 2 O ATP made NADPH made Principle Pathways Glycolysis TCA Cycle Electron Transport Oxidative Phosphorylation Pentose Phosphate Pathway ANABOLISM - Biosynthetic Rxns ATP consumed NADPH consumed MACROMOLECULE SYNTHESIS AND GROWTH ATP consumed, although sometimes other NTPs GTP-protein synthesis CTP-phospholipid synthesis UTP-polysaccharide synthesis ATP + NDP  ADP + NTP, so ATP is the ultimate source of these other NTPs Pathways are connected by a limited number of intermediates. Given the diversity of products of anabolism, it is surprising that the connection to catabolism is via so few molecules. Add PHOTOSYNTHESIS last – only in certain organisms, the phototrophs.

Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 6 ATP and NADPH are unique here. Intermediates are consumed by catabolism and replenished by anabolism. ATP and NADH are recycled (generally). They are unique in providing the link between catabolism and anabolism. Their function is coupling in both processes. ATP Stoichiometries Virtually every metabolic pathway makes or consumes ATP. In every case- the total reaction sequence is energetically favorable because of the ATP stoichiometry that accompanies it. Exergonic sequences are coupled to ATP synthesis. Endergonic sequences (by themselves endergonic) are rendered exergonic by coupling to ATP hydrolysis. Three types of stoichiometries : 1. Reaction stoichiometry C 6 H 12 O 6 + 6O 2 → 6CO 2 + 6PH 2 O respiration Here we account for each atom going in and out. Set by the laws of chemistry and predictable. 6 carbons going in, 6 carbons coming out, 18 oxygens in 18 oxygens out, etc. 2. Obligate Coupling Stoichiometry Respiration involves oxidation-reduction There is an obligate coupling of e- carriers, fixed by the chemistry of the overall process. (1) In glycolysis: C 6 H 12 O 6 + 10 NAD + + 2 [FAD + ] + 6 H 2 O → CO 2 + 10 NADH + 10 H + + 2 [FADH 2 ] (2) In the ETC: 10 NADH + 10 H + + 2 [FADH 2 ] + 6 O 2 → 12 H 2 O+ 10 NAD + + 2 [FAD + ] TOTAL : C 6 H 12 O 6 + 6 O 2 → 6 CO 2 + 6 H 2 O Because of the chemistry, could predict the nature of reactions (1) and (2), and their e- stoichiometries.

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Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 7 3. Evolved coupling stoichiometries The coupling to ATP synthesis here is fundamentally different. It is not fixed by the chemistry of the rxn sequence. It is limited by the energetics, but not fixed. Could not predict by examining the chemistry what the glucose to ATP stoichiometry would be. C 6 H 12 O 6 + 6 O 2 +38 ATP +38 P i → 6 CO 2 + 38 ATP + 44 H 2 O The number 38 is a consequence of the evolution of the system, which means a compromise between competing factors. What factors? The need to make ATP vs. the need to have the total  G negative enough to draw glucose oxidation to completion. The involvement of ATP serves to make unfavorable reactions favorable, to drive them forward, that is to change the equilibrium constant in favor of products. Because ATP + H 2 O  ADP + P i has a large –  G (-30.5kJ/mol), the equilibrium position has very low ATP and very high ADP & P i . But this reaction runs far from equilibrium. Kinetic controls over the rates of metabolic pathways are designed to ensure that [ATP]/[ADP][P i ] is very high. Why? So the ATP is available to provide driving force for all anabolic reactions. ATP and its derivatives have an additional role beyond driving endergonic reactions. It is an allosteric effector of enzymes sitting at key points in metabolism. For instance, ATP is a negative regulator of phosphofructokinase and pyruvate kinase in glycolysis, whereas AMP is a positive effector of phosphofructokinase and a negative regulator of FBPhosphatase.. Quantification of the Energy Status of a Cell We have said “something responds to conditions of high [ATP]…” What does that mean, exactly? Can that be quantified? The adenylate system is comprised of ATP, ADP and AMP. Their concentrations regulate almost all of metabolism. Often it’s: ATP + X  ADP + X-P Sometimes it’s for instance: ATP + X  AMP + PPi This happens, for instance, in the first step of fatty acid breakdown by AcylCoA Synthetase Fatty acid + ATP + CoASH  AMP + PP i + fatty acid-acyl-coA Adenylate kinase links AMP to the adenylate system by ATP + AMP  2ADP ;  G  ’  0, K eq = 1.2 So all three, ATP, ADP & AMP, are important.

Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 8 The Energy Charge defines how fully the system is charged with high-energy phosphates E.C.= 1 2 × 2[𝐴𝑇𝑃]+[𝐴𝐷𝑃} {𝐴𝑇𝑃]+[𝐴𝐷𝑃]+[𝐴𝑀𝑃] In English- The denominator is all the adenylate compounds present. The numerator is the number of phosphoric anhydride bonds available for chemical work. (Each phosphate bond in ATP and ADP are energetically equivalent.) The factor ½ normalizes the E.C. between 0 and 1. Since they are all linked, how does the concentration of each species vary as compared to the others? So cells maintain ATP at relatively high levels. Key regulatory enzymes respond in reciprocal ways to adenine nucleotides. I have mentioned a number of times that the activity of Phosphofructokinase is increased by AMP and decreased by ATP. In general, regulatory enzymes participating in energy- producing catabolic pathways show greater activity at low E.C., which drops sharply at E.C. approaching 1. In contrast, regulatory enzymes in anabolic pathways have low activity at low E.C., which rises sharply as EC approaches 1. These responses are called R (for ATP regenerating) and U (for ATP utilizing).

Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 9 PFK and pyruvate kinase are both R-responders Note that this is true for PFK even though PFK is itself an ATP-utilizing enzyme. Fructose-1,6-Bisphosphatase, which converts FBP to F-6-P in gluconeogenesis, is inhibited by AMP. Follows U response. Look at control over pathways exerted by the EC according to this graph. U and R pathways oppose each other with respect to ATP involvement. When the EC climbs above intersection point (meaning lots of ATP), then R declines precipitously and U increases sharply. This brings ATP down. When it crosses again, R goes up, U goes down, and ATP goes up. So at high ATP = EC → 1, R  , U  →  ATP =  EC, R  U  →  ATP… So the energy charge is driven to oscillate in steady state near the crossover point. In normal cells, EC~0.85-0.88. Because the EC is maintained at a steady state value, it is not such a useful index to determine the capacity of a cell to carry out phosphorylation reactions. Its utility is to understand how and why the [ATP] is held at a particular concentration. A better index of a cell’s capacity to run phosphorylation reactions is the PHOSPHORYLATION POTENTIAL. Look at ADP + Pi  ATP + H 2 O The phosphorylation potential is  = [ATP]/[ADP] [Pi]

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Biological Sciences 105 Lecture 17, December 5, 2017 Copyright Steven M. Theg, 2017. All federal and state copyrights reserved for all original material presented in this course through any medium, including lecture or print. 10  varies in cells more than does EC, with values between 200-800 M -1 , with higher levels meaning more ATP and higher potential to phosphorylate different substrates. The overall energy balance in a cell is regulated by AMP-activated protein kinase = AMPK AMPK is activated more than 1000-fold by AMP, meaning cellular energy levels are low. Once activated, it phosphorylates many of the enzymes that control key steps in energy consumption and production, turning them on and off as appropriate. ATP binds to the AMP binding site in AMPK, displacing the AMP and thereby inactivating the enzyme. Without this kinase activity, the enzyme targets are dephosphorylated, and so revert to the dephosphorylated activities. Which enzymes are targeted by AMPK? Phosphorylation by AMPK… stimulates Phosphofructokinase 2, which makes fructose - 1, 2 – bisphosphate, which stimulates glycolysis. Down-regulates glycogen synthase Down-regulates AcetylCoA carboxylase (fatty acid synthesis) Down-regulates HMG-CoA-reductase (cholesterol synthesis) AMPK also phosphorylates numerous transcription factors, leading to decreased transcription of genes encoding biosynthetic enzymes and increased expression of enzyme involved in catabolic reaction. So AMPK is referred to as the cell’s energy sensor.