BMB 462 Lecture 15 Outline of Last Lecture I. Continuation of the Urea CycleII. Connections between the Urea Cycle and the TCA CycleIII. Regulation of the Urea CycleIV. Metabolism of Carbon Skeletons from amino acidsV. Carbon TransfersVI. Phenylalanine DegradationVII. The Nitrogen CycleVIII. Nitrogen FixationOutline of Current Lecture I. Ammonia incorporation into Carbon CompoundsII. Amino Group TransfersIII. Amino Acid AnabolismIV. Important biomolecules derived from Amino acidsV. Hormonal SignalingVI. Hormone ClassificationCurrent LectureConcepts to remembers from previous courses/lectures:-I. Ammonia incorporation into Carbon Compoundsa. The system that moves Nitrogen around is called GS/GOGAT system. It’s the major route for incorporating Nitrogen.i. This is happening in plants. Humans can't incorporate free Nitrogen. 1. We get it already synthesized in Amino Acids from the protein in our diet.2. Plants and bacteria need to regulate the incorporation of Nitrogen.ii. Need glutamate to donate Nitrogen for aminotransferase.b. Glutamine Synthetase These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.i. Glutamate + NH4 + ATP Glutamine + ADP + Pi + H ii. ATP is the energy source. Glutamine is the nitrogen donor; nitrogen is then used for synthesis of other molecules.1. Enzyme that does this is amidotransferase iii. Removing the NH3 returns glutamine to glutamate so you can restart cycleand glutamine synthetase can pick up free ammonia and drop it off with transferase.iv. Aminotransferase uses glutamate as substrate.c. Glutamate Synthase – i. Glutamine + alpha-ketoglutarate + NADPH + H 2 glutamates + NADPii. Glutamine from above becomes substrate in synthase reaction.iii. Transfers gamma C from side chain to alpha ketoglutarate, which then provides two glutamates.1. Requires reducing power so NADPH is source of e-2. Now have 2 glutamates to use in aminotransferase reactions. One is source for regenerating glutamine.a. The other one goes down to aminotransferase and donatesan amino group for synthesis. When you drop it off, regenerates alphaketoglutarate (acceptor)d. Glutamate Dehydrogenasei. alpha-ketoglutarate + NH4 + NADPH + H Glutamate + NADP + H2Oii. Uses alphaketoglutarate and free ammonia + reducing power to get glutamate, which can then serve as glutamate used in generating glutamine.iii. Humans use glutamate dehydrogenase to run towards alpha-ketoglutarate and release free ammonia in the liver for urea cycle. In plants the process can run to generate glutamate to incorporate free Nitrogen. 1. This is a fairly minor route in the GDH/GS system.e. Regulation of Ammonia Incorporation – regulation in E. colii. 3 levels of regulation1. Allosteric - based on quick needs in the cell, regulated by levels of products. 2. Controlling enzymes already there by covalent modification3. Producing more enzymes.ii. 2 parts to allosteric regulation in bacteria: 1. Levels of glycine and alanine tell cell whether you have enough Amino Acids. When levels start increasing, there's going to be allosteric inhibition of glutamine synthetase2. Inhibition of the acceptors for amidotransferase; Glutamine is the e- donor. As glutamine increases, the levels of synthetase reduce.iii. The cell is incorporating just enough nitrogen to fulfill functions without wasting Energy on gathering excess.iv. Covalent Regulation of Glutamine synthetase1. Adenylylation: Adding AMP to enzyme (analogous to phosphorylation and dephosphorylation)2. Uridylylation is adding UMP.a. Both used to control various proteins 3. An indicator that there is enough Nitrogen is plenty of glutamine and lots of phosphate a. Means you broke down a lot of ATP, so don't have much energy left b. Don't want to be undergoing biosynthesis so glutamine synthesis gets an AMP added and becomes inactive.4. Layering this adenylylation with allosteric control is very sensitive regulation.v. Transcriptional Regulation1. When PII has UMP attached (activating glutamine synthetase), it also activates transcription of the GS gene. a. So the cell makes more of the protein as well. More protein is more activity.II. Amino Group Transfersa. Aminotransferases - use the alpha amino group from glutamate and move it to the alpha-keto acid.b. Amidotransferases - use side chain gamma amino group from glutamine; transferit to activated hydroxyl or a keto group.i. Donate Nitrogen to one or the other acceptor so that the product has Nitrogen incorporated.III. Amino Acid Anabolisma. Synthesis is Ketone groups to Amino Acids. Breakdown is Amino Acids to ketone groups.b. Anabolism is not simply the reverse of catabolism. The carbon is not coming fromthe same place it was broken down to. i. Some reactions in common but also have unique stepsc. Diverging pathways - start with a few intermediates (7 of them) and from them you make all 20 Amino Acids, so there has to be branches in pathway to make all of the Amino Acids.d. General characteristicsi. Take carbon from central metabolismii. Modify the carbon skeletons to get alpha-keto acids.1. This uses a lot of ATP for activation in order to do biosynthesis.2. Perform reduction of carbon, which will frequently require NADPHas e- donor. Many reactions also require PLP (this allows us to do chemistry around the alpha, beta, and gamma carbons of amino acids)iii. After you get alpha-keto acid, add an amino group using an aminotransferase. Use glutamate as a donor and move it to a keto group in alpha-keto acid. iv. Additional modifications are then performed to get to the correct final amino acid product.e. Sources of Carbon Skeletonsi. The core sources of carbon are from pentose phosphate pathway. 1. The ribose- and erythrose- phosphatesii. Glycolysis - 3phosphoglycerate, phosphoenolpyruvate, and pyruvateiii. TCA – alpha-ketoglutarate and oxaloacetate. 1. Only these 2, whereas breakdown had almost all intermediates going to TCA Cyclef. Transamination - aminotransferases add N.g. Biosynthetic Familiesi. Know precursors (alpha-ketoglutarate and oxaloacetate from TCA, pyruvate, 3PG, PEP from glycolysis, pentose phosphates)ii. Keto, amino acid pairs: Alphaketoglutarate and glutamate, oxaloacetate and aspartate, pyruvate and alanineh. Essential vs. Non-essential amino acidsi. Non-essential – the body can make enough of the amino acid that you don't need to include it in the dietii. Essential – The body
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