Presentation on theme: "Amino Acid Oxidation and the Production of Urea"— Presentation transcript:
1 Amino Acid Oxidation and the Production of Urea Chapter 18Amino Acid Oxidation and the Production of UreaAgain, in the Mitochondria where fatty acids are oxidized, amino acids are also oxidtized.
2 Amino Acid Oxidation, Production of Urea Key topics: To KnowHow proteins are digested in animalsHow amino acids are degraded in animals as a source of energyHow urea is made and excretedSome genetic defects in amino acid recycling
3 The use of amino acids as fuel varies greatly by organism About 90% of energy needs of carnivores can be met by amino acids immediately after a mealMicroorganisms scavenge amino acids from their environment for fuel when neededOnly a small fraction of energy needs of herbivores are met by amino acidsPlants do not use amino acids as a fuel source, but can degrade amino acids to form other metabolites
4 Metabolic Circumstances of Amino Acid Oxidation Leftover amino acids from normal protein turnoverDietary amino acids that exceed body’s protein synthesis needsProteins in the body can be broken down to supply amino acids for energy when carbohydrates and fats are scarce (starvation, diabetes mellitus)
5 Overview Intracellular protein turnover…remember the Proteasome. Dietary protein…remember the digestive endoproteases and exoproteases amino acids and small peptides are taken up by the intestinal epithelium.Amino acid degradation to CAC intermediates AND acetyl-S-CoA (not in the figure).Important point, amino acid nitrogen becomes in excess when amino acids are utilized + oxidized, therefore must be excreted (ammonium, uric acid, urea… in a safe form)
6 Proteosome Function Ubiquitin Binding Sites top and bottom Core ProteasomeThe Review continuesUbiquitin Binding Sites top and bottomNature 445:618 Feb 8, 2007
7 Ubiquitin Targeting a Cytoplasmic Protein 5th Edition: See pages , Fig 27-47, -486th Edition: See pages , Fig 27-47, -48.Protein Amino Terminal-aa Determines Protein’s Half-lifestabilizingM, G, A, S, T, V >20 hrsdestabilizingI, N, Y, D, P, L, F, D, K, R – 2 minReview of Ubiquitin tagging proteins for Proeasome activity.Proteasome peptidases are endopeptidases producing small peptides that diffuse away from the proteasome. These are cleaved by cytoplasmic amino-peptidases (exopeptidaes): these small peptides only last for a few seconds before they are converted to amino acids.Lets go first to getting rid of nitrogen…nitrogen processing to excretion.
8 Nitrogen Delivered to Liver Cells Again, the liver playing a major roll in metabolism: nitrogen processing in animals. All other tissues send amino acids to the liver in the form of glutamine (all tissues), alanine (muscles) and all dietary amino acids (intestine). Remember from General Biology: blood circulation from the intestine goes directly to the liver (hepatic portal vein, check it on in Wiki) first.
9 Nitrogen Excretion Forms A little review of General Biology: nitrogen excretion forms. Ammonia is a base and interacts with water to form the cation and OH-. Only organisms that can flush the surface that releases this can excrete ammonia: microbes, gilled animals, larvae. Birds and reptiles excrete uric acid a fully keto purine (and a breakdown product from diets with an excess of nucleic acids). This is largely insoluble so that is has few waters of hydration and is therefore light (particularly important for birds). Most vertebrates and sharks excrete urea: totally neutral (thus chemically safe), but exceptionally soluble. So what we will do first, is get amino acids to the liver and then make urea in the liver for delivery by blood through the kidneys to the urinary bladder.
10 A little review: pancreas secreters zymogens of the proteases A little review: pancreas secreters zymogens of the proteases. Major proteases in Small Intestine are trypsin, chymotrypsin, and carboxypeptidases. Both endo- and exo-proteases.
11 Transaminases EOC Problem 1 a Transamination Problem. Many amino transferases help getting amino to the liver, the chemistry is simple : amino keto and vitamin B6, pyridoxal phosphate, is the coenzyme (next slide).
13 Transaminases Used in Diagnosis of Tissue Damage Tissue damage leaks enzymes into blood(S)GPT: glutamate-pyruvate transaminase(S)GOT: glutamate-oxaloacetate transaminase-- indicate probable liver damage (toxins, infections)(S)CK: creatine kinase-- heart damage heart attack, infectionS for “serum”Here is a medically important clinical chemistry point: any tissue damage leaks enzymes into blood. Part of the Clinical Chem lab in a hospital is set up to measure transaminases. Leaking in to blood of SGPT or SGOT indicates liver damage, leaking SCK (creatinine kinase which is not a transaminase, indicates heart or other muscle damage. These assays are all now automated and easy to get done quickly from a blood sample. Lets get out of blood and into getting amino acids to the liver.EOC Problem 2: Measuring transaminases in blood – coupled reactions! (to things you already know)
14 Glutamate Dehydrogenase One of the early enzymes that made ammonium was Glutamate DH (E-DH), an oxidative enzyme producing ammonium and NAD(P)H. A process in amonotelic organisms.
15 Glutamine Synthetase This is just one reaction: Glutamine Synthetase Most organisms can convert ammonium to glutamine by glutamine synthetase. The figure shows the intermediate in the reaction, the reaction simply is E + ATP + NH4+ Q + ADP + Pi. Now the Q can be transported to the liver and converted to E and urea.
16 Glucose-Alanine Cycle is in Muscle Only EOC Problem 3: Alanine and Glutamine in Blood.The Glucose Alanine Cycle from muscle, uses A to transport nitrogen from muscle to liver (as well as E itself). When alanine enters the liver cells it gives up it nitrogen and becomes pyruvate (amino keto) which through gluconeogenesis can be converted to glucose and put into blood for the muscles (and rest of the body). Now lets to the Urea Cycle.
17 Urea Cycle OverviewAmino acids come from recycling (glutamine), muscle glycolysis (alanine), and diet.The Urea Cycle was worked out by that great cycle scientist, Hans Krebs. In fact he did this before the CAC ! The cycle takes part in two places: mitochondria and cytoplasm (where the urea is spun-off). The beginning of nitrogen processing is in the mitochondria, next slide.
18 Begin in the Mitochondrion From blood amino acids (diet) and glutamine – alanine from tissues enter as glutamine or all others get transaminated to get the nitrogen onto E. Both E and Q are transported into the mitochondion, and Q is hydrolysed (glutaminase) to E + NH4+. The E from other amino acids and gutamine produces NH4+ by E-DH. E is also the source of nitrogen to make D by tansaminase with OAA producing D and α-KG. Remember this is the mitochondria and both OAA and α-KG are part of CAC.Now the reaction to form the first entry of nitrogen to the Urea Cycle is the formation of carbamoyl-P from HCO3-, 2 ATP and NH4+. You can see the carbamoyl-P structure has atoms from all three substrates…and will be a substrate, with ornithine (a non protein amino acid, in Chapter 3) to form citrulline by the enzyme ornithine transcarbamoylase. Look at the structure of ornithine it looks just like lysine (K) but has one less –CH2- (remember K is an ε-amino and Ornithine is δ-amino). Lets move now to the cytoplasm.
19 The second reaction takes citrulline and an ATP + D to make argininosuccinate. The figure shows the intermediate only to emphasize that when using ATP it is breaking the bond between the alpha and beta phosphates (just like DNA polymerase, Chapter 8) producing Ppi and hooking the D amino group to the keto end of citrulline to form a quanido (three nitrogens on a carbon) group. This metabolite gets it’s name from the fact that the carbon part of aspartate looks a lot like succinate.Argininosuccinate is then cleaved by argininosuccinase to form fumarate and arginine the substrate of argininase (look, these two enzymes have sort of similar names) with hydrolyses arginine to ornithine and urea.OK…these reactions are in the cytoplasm, so what happens to the fumarate (a CAC intermediate) is that it is converted by fumarase to malate (which itself can be recycled into the mitochondria (see below for malate-aspartate shunt, but called aspartate-argininosuccinate shunt in your text) or malate DH coverts the malate to OAA. But these are CAC enzymes and not in the mitochondria where CAC is. They are cytoplasmic forms of the CAC enzymes and form OAA so that it can be as substrate for gluconeogenesis. OMG ! The connections are amazing !OVERALL: Urea cycle has two inputs of nitrogen, one input of one carbon, and 3 ATPs driving the cycle. Do the work to see that this is right or wrong:NH4+ + HCO3- + D + 3 ATP urea + fumarate + 2ADP + AMP + 2Pi + PPi Next slide to review the two nitrogen inputs.
20 Mechanims of the Two Nitrogen Entry Points in Urea Cycle Nitrogen-acquiring reactions in the synthesis of urea. The urea nitrogens are acquired in two reactions, each requiring ATP. (a) In the reaction catalyzed by carbamoyl phosphate synthetase I, the first nitrogen enters from ammonia. The terminal phosphate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate. In other words, this reaction has two activation steps (1 and 3). (b) In the reaction catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. Activation of the ureido oxygen of citrulline in step 1 sets up the addition of aspartate in step 2.
21 Relationship to Citric Acid Cycle EOC Problem 4: Alanine and Lactate have identical oxid-state, why do cells get less energy from alanine?EOC Problem 8: Asp Transaminase activitiy.While some of the malate in the cytoplasm can go to gluconeogenesis (by way of OAA), the malate aspartate shunt (here called aspartate-argininosuccinate shunt). In any event malate can enter the mictochondria and get converted to OAA with gets transaminated by E to form D and α=KG.
22 Urea Cycle Controlled Acetyl-Glutamate Acetyl-Glu is in the Arg synthetic pathway in bacteria and plants, in animals it is only regulatoryThis is of interest only (no exam questions), N-acetylglutamate synthase is the only surviving (through evolution from prokaryotes of an arginine biosynthetic pathway) which is used in animals only as a regulatory system to sense the amount of R in the cell…so that then N-acetylglutamate is formed as an allosteric effector. This effector signals that the cell is loaded with R as a signal that all amino acids are present so that carbamoyl phosphate synthetase is activated and begins to drive the urea cycle…to get rid of the amino nitrogens so the amino acid carbons can be used as a source of energy and gluconeogenesis.
23 Nonessential amino acids are those that we, as animals, have the complete biosynthetic pathway to produce our own aa’s from metabolic intermediates. The essential aa’s represent aa biosynthetic pathways we have lost during our evolution. We get plenty of these amino acids in our diets. Conditionally essential are those that we have the biosynthetic pathways, but they might not be producing enough of those amino acids under certain condtions.
24 Benzoate and Phenylbutyrate Given to Lower Blood Ammonium Treatment for genetic defects in Urea CycleExcreted in UrineA medical aspect of people who have defects in their urea cycle: they have an elevated level of ammonium in blood, giving benzoic acid or phenyl-butyrate will interact with some enzymes to produce product that are excreted by the kidneys thereby lowering blood ammonium.
25 Summary of Amino Acid Catabolism FIGURE 18–15 Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to different end products. The figure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, for instance, is degraded via at least two different pathways (see Figs 18–19, 18–27), and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the figure, by color shading. Notice that five of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, leucine and lysine, are exclusively ketogenic.
26 1 Carbon Transfer Cofactors There are many pathways here, so lets just focus on some key reactions that are found in many of these pathways. Firstly are the transaminases to get their nitrogen off to excretion.Secondly, there are many one-carbon transfers that use different coenzymes, those in the figure. Biotin in involved in carboxyl transfers (we have done this already in gluconeogenesis in converting pyruvate to OAA, pyruvate carboxylase). S-adenosylmethionine transfers the methyl attached to the sulfur. And, tetrahydrofolate can transfer carbonyls, methylenes and methyls and some others, it’s pretty versatile.Thirdly there are dehydrogenases. And, then a lot of other enzymes…to convert the aa’s to CAC intermediates or acetyl-S-CoA. Lets “just look” at some of these pathways.
28 Glycine Degradation in Kidney This is the source of the material in kidney stones.D-amino acid oxidase breaks down D-aa’s from bacterial peptidoglycan.Oxalate-Ca++ are major substance in kidney stones.
31 Phenylalanine Degradation Genetic defects in phenylalanine degradation. Lacking a functional phenylalanine hydroxylase can result in Phenyl-Keto-Urea, a condition that could result if life long mental retardation.
32 PKU = Phenylketouria EOC Problem 11: Analysis of blood and PKU Phenylkeouria results from serum phenylalanine being converted to phenylpyruvate (hence the “keto” uria) which can for phenylacetate and phenyllactate. Babies today are immediately tested for this just after birth. If they are PKU babies, they have to be on diet low in phenylalanine to for the first 5-10 years of life so that they do not become mentally retarded. This was one of the first genetic diseases also called “inborne errors in metabolism”.
36 Amino Acid Carbon UseFIGURE Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to different end products. The figure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, for instance, is degraded via at least two different pathways (see Figure 18-19, 18-27), and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the figure, by color shading. Notice that five of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, leucine and lysine, are exclusively ketogenic.
37 TABLE 18-2 Some Human Genetic Disorders Affecting Amino Acid Catabolism
38 Things to Know and Do Before Class Amino acids from protein are an important energy source in carnivorous animals and during starvation.2. The first step of AA catabolism is transfer of the NH3 via PLP-dependent aminotransferase usually to - ketoglutarate to yield L-glutamate.3. In most mammals, toxic ammonia is quickly converted to carbamoyl phosphate and passed into the urea cycle4. Amino acids are degraded to pyruvate, acetyl-CoA, α- ketoglutarate, succinyl-CoA, and/or oxaloacetate5. Amino acids yielding acetyl-CoA are ketogenic.6. Amino acids yielding other end products are glucogenic.Genetic defects in amino degradation pathways result in a number of human diseases: our example is PKU.EOC Problems 1-5, 8, 11.