MSBS 501 Biochemistry & Cell Biology 25 - Urea Cycle PDF

Copyright Notice and Agreement Class document distributed for the exclusive use of students in the CUSOM, MSBS class. Student access to and use of class materials are conditioned on agreement with the terms and conditions set out below. Any student who does not agree to them is prohibited fr...

Copyright Notice and Agreement Class document distributed for the exclusive use of students in the CUSOM, MSBS class. Student access to and use of class materials are conditioned on agreement with the terms and conditions set out below. Any student who does not agree to them is prohibited from accessing or making any use of such recordings. Any student accessing class materials (1) acknowledges the faculty members’ intellectual property rights in lectures and class materials and that distribution violates the CUSOM Copyright Policy; (2) recognizes the privacy rights of fellow students who speak in class; (3) accepts that distributing, posting, or uploading class materials to students or any other third party not authorized to receive them or to those outside CUSOM is an Honor Code violation; and (4) agrees that all class materials are to be Urea Cycle Jackson T. Sparks, PhD – Smith Hall 437 [email protected] -- 910-814-4949 MSBS 501 2024  Learning Objectives – Urea Cycle Name the ways amino acids are added or removed from the pool to achieve or alter nitrogen balance in the body Describe the process of protein digestion in terms of where it takes place and the sequence of events accounting for the following terms: pepsinogen, pepsin, zymogen, cholecystokinin, secretin, enteropeptidase, trypsinogen, trypsin, chymotrypsinogen, chymotrypsin, endopeptidases, exopeptidases Name the amino acids associated with the following carbon skeletons: alpha-ketoglutarate (2 amino acids), pyruvate (1), and oxaloacetate (1) Describe how nitrogen can be added, removed, or transferred between different carbon skeletons using aminotransferases, glutamate dehydrogenase, glutamine synthetase, and glutaminase Describe the urea cycle in terms of its purpose, how it is regulated, what physiological circumstances would increase or decrease the overall activity, what can cause a defect and what will be the consequence of a defect, and the contribution of the following: ammonia, carbon dioxide, ornithine, aspartate, arginine, carbamoyl phosphate synthetase I (CPS I), ornithine transcarbamoylase (OTC), and arginase Describe the role of the liver and kidney in terms of urea and ammonia as to production and elimination of nitrogen from the body Explain how urea cycle defects cause hyperammonemia, and use the knowledge of nitrogen metabolism to suggest ways in which the symptoms could be attenuated  Nitrogen Metabolism Nitrogen metabolism will include: Amino Acid and Protein metabolism Heme Metabolism Nucleotide Metabolism One important point when considering nitrogen metabolism vs carbohydrate metabolism or fat (lipid) metabolism, is that humans have no storage form of nitrogen, ie, there is no protein that exists simply to store amino acids (whereas we store glucose (glycogen) and lipids (TAGS, cholesterol)).  Nitrogen Metabolism Another point to consider, is that nitrogen itself is a reactive compound (most explosives contain nitrogen!) and so, the body must get rid of any nitrogen that is above the needs for any nitrogen containing compounds. Most nitrogen will leave the body via the Urea Cycle, which serves to rid the body of excess nitrogen, but also retaining any “carbon skeletons” of the nitrogen containing compounds for other uses.  Nitrogen Metabolism Most nitrogen enters the body in the form of amino acids, so we can think of the overall big picture of nitrogen metabolism by considering the “amino acid pool” in the body. So, we have three contributions adding to the pool: 1) amino acids from dietary protein 2) amino acids from protein turnover in the body 3) synthesis of non-essential amino acids (non-essential amino acids are those we do not have to get from the diet, ie we can synthesize from other precursors. Conversely, essential amino acids are those we can not synthesize, or those we can not synthesize enough of).  Nitrogen Metabolism And three way of amino acids being removed from the pool: 1) synthesis of proteins 2) synthesis of other nitrogen containing compounds (for example nucleotides and heme) 3) use of the “carbon skeleton” of amino acids for other compounds (ie, glucose, lipids, ketone bodies), or for energy. In these scenarios we have to get rid of the nitrogen. The term “nitrogen balance” that is often used in nutrition is referring to the balance, net loss, or net gain of amino acids in the “pool”  Protein Digestion Nitrogen Metabolism Protein must be broken down into amino acids or small peptides before they can be absorbed. Stomach – low pH helps denature proteins (main purpose of low pH is to kill microorganisms, secondary effect is a digestion aid). Pepsin, an endopeptidase, is the main enzyme for protein digestion in the stomach, secreted by cells as a zymogen (inactive precursor) pepsinogen that is cleaved to its active form by the low pH of the stomach. Activated pepsin can also further activate other pepsinogen molecules.  Protein Digestion Nitrogen Metabolism Small intestine – Large polypeptides enter the small intestine, and further digestion takes place by enzymes from the pancreas. Cholecystokinin and secretin are hormones that stimulate the pancreatic secretion. There is a cascade of events that occur to release the active digestive enzymes from their zymogens.  Protein Digestion Nitrogen Metabolism There is a cascade of events that occur to release the active digestive enzymes from their zymogens. 1) Enteropeptidase – synthesized and present on luminal surface of intestinal mucosal cells, cleaves trypsinogen to yield trypsin. 2) Trypsin further affects its own release by cleaving trypsinogen to trypsin. 3) Trypsin cleaves other pancreatic zymogens (for example chymotrypsinogen) to yield activated enzymes (for example, chymotrypsin).  Protein Digestion Nitrogen Metabolism All the enzymes contribute different activities to digest the proteins. The enzymes cleave specific sequences of amino acids and may be endopeptidases or exopeptidases (so may cleave INSIDE (endo) a peptide sequence or from the ENDS (exo) of a peptide sequence.) Eventually, only free amino acids are released into the portal vein. As with much of metabolism, the liver plays a big role, as it gets first shot at absorbing amino acids and determines which amino acids will be released into general circulation, and how much.  Removal of Nitrogen and Transport A key to understanding the movement of nitrogen through the body, is understanding the use of common carbon skeletons that will link the removal of nitrogen from all amino acids to just a few key compounds, using carbon skeletons you are already familiar with, from other sections of metabolism. If you learn these few, it makes the whole section much simpler and really gives you a good feel for how amino acid metabolism fits into other areas of metabolism.  Removal of Nitrogen and Transport -ketoglutarate + NH3 = Glutamate Glutamate + NH3 = Glutamine Pyruvate + NH3 = Alanine Oxaloacetate + NH3 = Aspartate You have seen -ketoglutarate and oxaloacetate as intermediates in the TCA cycle, and pyruvate as an end product of glycolysis.  Removal of Nitrogen and Transport The majority of nitrogen transfers in metabolism involve the -ketoglutarate skeleton. There are a number of aminotransferases that will catalyze the transfer of the amino group between the amino acid and -ketoglutarate, or the opposite reaction from glutamate to the - keto acid skeleton of the amino acid. There are different aminotransferase enzymes for most of the amino acids. All aminotransferases require the coenzyme pyridoxal phosphate (a vitamin B6 derivative).  Removal of Nitrogen and Transport The two most important aminotransferases in humans are alanine aminotransferase (ALT) and aspartatate aminotransferase (AST). These two enzymes also serve another important function in medicine, as indicators of tissue damage when found in the bloodstream for example in liver failure or a heart attack.  Removal of Nitrogen and Transport Glutamate dehydrogenase – catalyzes the removal of amino group from glutamate to yield free ammonia and - ketoglutarate (or vice versa). In this case ammonia is a product (or substrate) in the reaction instead of transferring between different amino acids. Primarily located in liver and kidney (also shown in next figure in muscle).  Removal of Nitrogen and Transport Glutamine synthetase – Addition of free ammonia to glutamate to make glutamine. This occurs in many tissues, and is the form in which these tissues release nitrogen into the bloodstream for transport to the liver Glutaminase – this enzyme performs the opposite reaction as glutamine synthetase, releasing free ammonia and glutamate in the liver.  Removal of Nitrogen and Transport So, the major routes of nitrogen disposal to the liver occur from most tissues as glutamine released into the bloodstream and taken up by the liver to be used in the urea cycle. An additional mechanism is used mainly by muscle tissue in which the amino group is part of alanine, which can be made using pyruvate (as an end product of glycolysis in muscle).  Removal of Nitrogen and Transport So, alanine and glutamine are the major amino acids released into the bloodstream for transport to the liver for disposal of nitrogen. The carbon skeletons of each of these (pyruvate or -ketoglutarate) can easily be fed into other biosynthetic pathways while the nitrogen will be disposed of using the urea cycle (as well as a small amount of free ammonia).  Urea Cycle Urea will be the form in which about 90% of the nitrogen for disposal is removed from the body (~10% ammonia, ~1% uric acid). Urea is formed by the actions of the urea cycle in the liver. Important points to know about the urea cycle:  Urea Cycle 1) Look at the final structure of urea in the figure. The carbon and oxygen come from carbon dioxide (so no net loss of carbon) and the amino groups come from free ammonia and aspartate. 2) When looking at the intermediates, you will notice the carbon skeletons in the cycle that are used as carriers (for example ornithine) are not lost but regenerated as the cycle is completed.  Urea Cycle 3) The first reaction is catalyzed by Carbomyl Phosphate Synthetase I (CPS-I), which uses carbon dioxide, ammonia and ATP to form carbomoyl-phosphate. This is the rate-limiting and regulated step for the urea cycle. (Note, there will be a CPS-II in a later section on nitrogen metabolism, make sure you remember which is which!)  Urea Cycle 4) The second reaction is catalyzed by ornithine transcarbomylase which takes ornithine and carbomyl phosphate and makes citrulline. A deficiency in this enzyme is the most common urea cycle defect.  Urea Cycle 5) The first two reactions take place in the mitochondria, citrulline is than transported to the cytoplasm to complete the urea cycle. Following the completion of the cycle, ornithine will be regenerated, and transported back into the mitochondria to be used once again in another round of the urea cycle.  Urea Cycle 6) For the steps in the cytoplasm, note the following: a) the second amino group is added, which comes from aspartate b) notice there are common intermediates with the TCA cycle (malate, fumarate, and oxaloacetate) c) Arginase releases free urea  Urea Cycle The urea that is synthesized by the urea cycle will be released into the bloodstream, and filtered by the kidneys, where it will be excreted in the urine.  Urea Cycle Regulation of the urea cycle – CPS-I, the rate limiting step in the urea cycle, is activated by n-acetylglutamate. In turn, n-acetylglutamate synthase uses acetyl CoA and glutamate, and arginine is an activator, SO, overall we have: High acetyl-CoA + high glutamate + high arginine  n-acetylglutamate  activate CPS-I  Activate urea cycle  Urea Cycle High acetyl-CoA + high glutamate + high arginine  n-acetylglutamate  activate CPS-I  Activate urea cycle So, the urea cycle will be stimulated by a high protein meal! Additionally, through mechanisms not detailed here, a high protein diet will increase synthesis of urea cycle enzymes via increased transcription of the genes.  Movement of ammonia from peripheral tissues to the liver Any free ammonia present in the circulation will be removed by the liver and used for the synthesis of urea. A small amount of free ammonia is obtained in the diet from the action of microorganisms or endogenous enzymes in the intestine.  Movement of ammonia from peripheral tissues to the liver The kidneys themselves, in addition to removing urea from circulation via filtration, also produce some free ammonia, via glutaminase. This ammonia is released in the urine (~10% of the nitrogen in the urine is free ammonia) and contributes to the acid-base balance of the body by excreting protons (so its actually the ammonium ion, NH4+). This will be important later in physiology and pharmacology.  Hyperammonemia There are also diseases in which there are defects in an enzyme of the urea cycle, that will manifest as hyperammonemia (depending on which enzyme is defective, other products may accumulate as well). The most common is Ornithine Transcarbomylase Deficiency (OTC), an X-linked disorder with an estimated incidence of 1:20,000, although defects are possible in any of the enzymes (as well as transporters or effectors involved in the urea cycle). Urea cycle defects fall under a general category of inborn errors of metabolism. Most of these diseases, when considered alone are quite rare, but when all the different diseases are added up, inborn errors of metabolism are quite common (genetically speaking).  Hyperammonemia Urea cycle defects are, generally speaking, very dangerous and had a high death rate (the symptoms will manifest shortly after birth, and unless the hyperammonemia is brought under control quickly, irreversible damage will occur). Knowledge of metabolism has allowed the design of diets to essentially eliminate as much as possible the “need” to use the urea cycle (this will obviously take careful consideration of intake of protein).