Principles of Biochemistry Lecture 24 Spring 2024 PDF

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 24 Nitrogen Metabolism: Amino acid oxidation and the Urea Cycle Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 18: p. 625-639 AMINO ACID OXIDATION AND UREA PRODUCTION Key topics: – How proteins are digested in animals – How amino acids are degraded in animals – How urea is made and excreted METABOLIC CIRCUMSTANCES OF AMINO ACID OXIDATION amino acids undergo oxidative degradation when: – amino acids released during protein turnover are not needed for new protein synthesis – ingested amino acids exceed the body’s needs for protein synthesis – cellular proteins are used as fuel because carbohydrates are either unavailable (starvation) or not properly utilized (disease, i.e. diabetes) DIETARY PROTEINS ARE ENZYMATICALLY HYDROLYZED INTO AMINO ACIDS Pepsin degrades proteins into large peptides in the stomach Trypsin, chymotrypsin, elastase, aminopeptidase and carboxypeptidases degrades large peptides into smaller peptides and amino acids in the small intestine Amino acids and di- and tripeptides are transported (through transporters) into the intestine lining cells à blood à liver OVERVIEW OF AMINO ACID CATABOLISM used for § The amino groups and the carbon skeleton take separate but interconnected pathways: § The amino group enters the urea cycle and nitrogen is excreted in the form of urea excreted § The carbon skeleton enters the citric acid cycle to be oxidized METABOLIC FATES OF AMINO GROUPS AMINO GROUP CATABOLISM Ammonia is toxic Urea is far less toxic than ammonia and has very high solubility Uric acid is rather insoluble Excretion as paste allows the animals to conserve water unless re-used, amino groups are channeled into a single excretory end product: Humans and great apes excrete both urea (from amino acids) and uric acid (from purines) THE AMINO GROUP IS TRANSFERRED TO a-KETOGLUTARATE FORMING L-GLUTAMATE Urea or synthesis Catalyzed by aminotransferases or transaminases: Uses the pyridoxal phosphate (PLP) cofactor Typically, a-ketoglutarate accepts amino groups and is converted into glutamate Glutamate acts therefore as a temporary storage of nitrogen (in its amino group) Glutamate can then donate the amino group to form urea for amino acid biosynthesis when needed The amino acid is converted to an a-ketoacid TRANSAMINASES Cells contain many different Transaminases (aminotransferases). They are specific for α-ketoglutarate as the amino group acceptor, but differ in their specificity for the amino group donor. Named for the amino group donor: For instance, alanine aminotransferase will transfer an amino group from alanine to α-ketoglutarate to form pyruvate and glutamate. Aspartate aminotransferase will transfer an amino group from aspartic acid to α-ketoglutarate to form glutamate and oxaloacetate. The reactions catalyzed by transaminases are reversible. All amino transferases use pyridoxal phosphate (PLP) as a prosthetic group. This is the coenzyme form of pyridoxine, or vitamin B6. Pyridoxal phosphate is tightly bound to the transaminases active site through a Schiff-base (covalent) and other non-covalent interactions. PLP IS COVALENTLY LINKED TO TRANSAMINASES By an internal aldimine The linkage is made via a nucleophilic attack of the amino group of an active-site lysine PLP (red) bound to one of the two active sites of the dimeric enzyme aspartate aminotransferase, a typical aminotransferase STRUCTURE OF PLP AND PYRIDOXAMINE PHOSPHATE Intermediate, enzyme-bound carrier of amino groups Aldehyde (PLP) form can react reversibly with amino groups Aminated (pyridoxamine) form can react reversibly with carbonyl groups Pyridoxal phosphate, the prosthetic group of aminotransferases. (a) Pyridoxal phosphate (PLP) and its aminated form, pyridoxamine phosphate, are the tightly bound coenzymes of aminotransferases. The functional groups are shaded. PLP FUNCTIONS AS AN INTERMEDIATE CARRIER OF AMINO GROUPS AT THE ACTIVE SITE OF TRANSAMINASES PLP undergoes a reversible transformation between its internal aldimine form (pyridoxal phosphate) which can accept an amino group and its aminated form (pyridoxamine phosphate) which can donate its amino group to a keto acid, through a Shiff base intermediate (left to right). To complete the process, a second α-keto acid replaces the one that is released, and this is converted to an amino acid in a reversal of the reaction steps (right to left). Pyridoxal phosphate is also involved in certain reactions at the β and γ carbons of some amino acids (not shown) TRANSAMINASE LEVELS IN SERUM CAN INDICATE TISSUE DAMAGE - Transaminase (or amino-transferase levels) in human serum are often monitored to gain information concerning tissue damage for heart, liver and pancreas. - When tissues are damaged, the levels of a given enzyme may rise within the tissues and leak out into the bloodstream (then eventually removed). The time course of the rise and fall depends on the enzyme as well as the injury and the tissue. - The enzyme(s) selected as an indicator for tissue damage would ideally be specific for this tissues, ( i. e. an isoenzyme found only, or predominately, in that tissue). - The tests typically measure two transaminases, sGOT - serum glutamate-oxaloacetate transaminase-(also called aspartate aminotransferase, AST) and sGPT – serum glutamate-pyruvate transaminase – (also called alanine aminotransferase, ALT). IN THE LIVER, AMMONIA COLLECTED IN GLUTAMATE IS REMOVED BY GLUTAMATE DEHYDROGENASE (GDH) TO BE EXCRETED AS UREA In hepatocytes (Liver), Glutamate is transported from the cytosol to mitochondria and is deaminated by oxidative deamination within mitochondrial matrix, forming a-ketoglutarate GDH can use either NAD+ or NADP+ as electron acceptor Ammonia is processed into urea for excretion α-ketoglutarate can enter the citric acid cycle or be used for glucose synthesis Thus, the pathway for ammonia excretion is a transdeamination = transamination (transaminases) + oxidative deamination (GDH) Urea à excretion GLUTAMATE DEHYDROGENASE OPERATES AT AN IMPORTANT INTERSECTION IN CARBON AND NITROGEN METABOLISM α-ketoglutarate made by GDH reaction can be oxidized as fuel (in CAC) or serve as a glucose precursor in gluconeogenesis (through oxaloacetate) glutamate dehydrogenase is allosterically: – Activated by ADP (at low glucose or energy levels à AA degradation is increased àuse of carbon skeletons of AA for energy – Inhibited by GTP (at high levels of α-ketoglutarate) IN THE MUSCLE, AMMONIA COLLECTED IN GLUTAMATE IS DONATED TO PYRUVATE TO MAKE ALANINE Vigorously working muscles rely on glycolysis for energy: à yields pyruvate and operate nearly anaerobically if pyruvate is not eliminated lactic acid will build up To avoid lactic acid build-up, this pyruvate can be converted to alanine (by taking up the amino group from glutamate) for transport into liver: Once in the liver, alanine releases its amino group and leads to pyruvate, which is used to produce glucose. Glucose returns to the muscle and the ammonia is excreted (see the glucose-alanine cycle). ALANINE TRANSPORTS AMMONIA FROM SKELETAL MUSCLE TO THE LIVER: THE GLUCOSE-ALANINE CYCLE GDH § Alanine plays a special role in transporting amino groups to the liver via the glucose-alanine cycle. § In this cycle, the amino group of aminoacids collected in glutamate is transferred to pyruvate, using alanine aminotransferase (an alternative to the formation of glutamine via the glutamine synthetase reaction). § The alanine formed in this reaction can then be transported to the liver, where transamination once again produces pyruvate and glutamate § The pyruvate can then be used to synthesize glucose, via gluconeogenesis, which is exported back to the muscles. (Recall, lactic acid formed during vigorous activity is also exported to the liver for gluconeogenesis by the Cori Cycle). § The ammonia formed by deamination of glutamate (GDH reaction) is excreted. AMMONIA FROM OTHER TISSUES IS SAFELY TRANSPORTED TO THE LIVER IN THE BLOODSTREAM AS GLUTAMINE § Many tissues, including brain, generate free ammonia and in order to prevent the accumulation of high levels of this toxic substance, it must be converted into a non-toxic form for transport. § Excess ammonia in extrahepatic tissues is added to glutamate to form glutamine (non-toxic), a process catalyzed by glutamine synthetase (requires ATP). § After transport in the bloodstream, the glutamine enters the liver mitochondria where glutaminase, hydrolyzes the ammonia from the side chain of glutamine, converting back glutamine to glutamate. § NH4+ thus liberated is incorporated in urea then excreted (excess glutamine is processed in intestines, kidneys, and liver). excretion AMMONIA IS TOXIC NH3 crosses the blood brain barrier and accumulates in cells Free ammonia is toxic, especially to the brain (cognitive impairment, ataxia, epileptic seizures, swelling of brain and death in extreme cases) NH4+ competes with K+ for transport into astrocyte cells through Na+K+ ATPase – Leads to elevated extracellular [K+] in neurons à – Excess K+ enters through Na+-K+-2Cl- cotransporter 1 (NKCC1) , a symporter that transports K+, but also Na+, and Cl– excess Cl- from the excess K+ alters neuronal response to the neurotransmitter GABA à neuromuscular incoordination and seizures… THE FATE OF AMMONIA: THE UREA CYCLE AND NITROGEN EXCRETION HIGHLY TOXIC AMMONIA MUST BE UTILIZED OR EXCRETED IN UREA Urea contains two amino groups The first amino group of urea comes from free ammonia (released from glutamate or glutamine). This is done through Carbamoyl phosphate synthase I which captures the free ammonia in the mitochondrial matrix First step of the urea cycle This step is regulated The second amino group of urea is acquired from aspartate THE FIRST NITROGEN-ACQUIRING REACTION OF THE UREA CYCLE (Synthesis of carbamoyl phosphate from free ammonia) 1 1 This reaction uses 2 ATPs Nitrogen of carbamylphosphate enters the urea cycle In this first nitrogen-acquiring 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. This reaction has 2 activation steps (1 and 3). THE SECOND NITROGEN-ACQUIRING REACTION OF THE UREA CYCLE (Entry of aspartate into the urea cycle) This reaction uses 2 ATPs Nitrogen of arginino-succinate enters in urea composition In this second nitrogen-acquiring reaction, catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. Activation of the ureido oxygen of citrulline in step 1, using 1 ATP and releasing PPi, sets up the addition of aspartate in step 2. THE AMINO GROUP OF GLUTAMATE OR GLUTAMINE IS METABOLIZED IN THE MITOCHONDRIA OF HEPATOCYTES Free ammonia (NH4+) released from glutamate (or glutamine) is converted to urea for excretion. Carbamoyl phosphate synthase I captures free ammonia in the mitochondrial matrix First step of the urea cycle Regulated One amino group of urea (from glutamate or glutamine) enters the urea cycle as carbamoyl phosphate, formed in the matrix; the other enters as aspartate, formed in the matrix by transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase. Urea cycle reactions Urea THE REACTIONS IN THE UREA CYCLE Urea cycle and reactions that feed amino groups into the cycle. The enzymes catalyzing these reactions are distributed between the mitochondrial matrix and the cytosol. One amino group enters the urea cycle as carbamoyl phosphate, formed in the matrix; the other enters as aspartate, formed in the matrix by transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase. The urea cycle consists of four steps (3 are irreversible): 1 Formation of citrulline from ornithine and carbamoyl phosphate (entry of the first amino group); the citrulline passes into the cytosol (Ornitine Transcarbamylase) 2 Formation of argininosuccinate through a citrullyl-AMP intermediate (entry of the second amino group) (Arginosuccinate synthase). 3 Formation of arginine from argininosuccinate; this reaction releases fumarate, which enters the citric acid cycle (Arginosuccinase). Only reversible step. 4 Formation of urea; this reaction also regenerates ornithine (Arginase). ATP COST OF THE UREA CYCLE § Urea is produced from ammonia in five enzymatic steps and requires 4 molecules of ATP. § Two molecules are required to form carbamoyl phosphate and the equivalent of two further ones are required in the formation of argininosuccinate. (ATP to AMP + PPi then PPi to 2 Pi has the same energy cost as the hydrolysis of 2 molecules of ATP to ADP + Pi). to malate and CAC § Some of the energy cost is made up by converting fumarate to malate (see next slide). The rest comes from GDH reaction (see next slide) THE UREA CYCLE AND CITRIC ACID CYCLE ARE LINKED THROUGH THE ASPARTATE-ARGINOSUCCINATE SHUNT The interconnected, urea and citric acid, cycles have been called the "Krebs bicycle." NH4+ Glutamate Makes 2.5 ATP Glutamate DH reaction makes 2.5 ATP The aspartate-argininosuccinate shunt (linking the citric acid and urea cycles), effectively link the fates of the amino groups and the carbon skeletons of amino acids. § In converting fumarate to malate, then malate to oxaloacetate, one molecule of NADH is generated, whose re-oxidation through the oxidative phosphorylation process generates 2.5 ATP. Thus the energy cost of the urea cycle is 1.5 ATP. § However, the glutamate dehydrogenase reaction that gives the ammonia to the urea cycle (see above) leads to the formation of one NADH and thus to 2.5 ATP molecules. § Thus, the urea cycle can be considered as The interconnections are even more elaborate than the arrows suggest. For example, some citric acid self-sustaining cycle enzymes, such as fumarase and malate dehydrogenase, have both cytosolic and mitochondrial isozymes. Fumarate produced in the cytosol—whether by the urea cycle, purine biosynthesis, or other processes—can be converted to cytosolic malate, which is used in the cytosol or transported into mitochondria (via the malate-aspartate shuttle) to enter the citric acid cycle. REVIEW: FLOW OF NITROGEN FROM AMINO ACIDS TO UREA Overall stoichiometry of the urea cycle Aspartate + NH3 + HCO3- + 3 ATP + H20 à Urea + fumarate + 2 ADP + AMP + 2 Pi + PPi PPi à 2Pi (1 ATP equivalent): Thus, 4 ATP are used Urea is mainly excreted in the urine; some enters the intestine where bacterial urease cleaves it to CO2 and NH3 (which is lost in the feces and partly reabsorbed into the blood) When liver function is compromised or due to genetic defects in the urea cycle à Hyperammonemia (>>10 μM), NH3 is neurotoxic for reasons that are not clear but could include: - consumption of glutamate and ATP to make glutamine; - depletion of TCA cycle intermediate α-ketoglutarate to make glutamate; - neurotransmitter function of glutamate à GABA REGULATION OF THE UREA CYCLE BY N-ACETYL-GLUTAMATE N-acetylglutamate is formed by Nacetylglutamate synthase – When glutamate and acetyl-CoA concentrations are high (indicating the presence of high levels of aminoacids) N-acetylglutamate activates carbamylphosphate synthetase I, thus activating the urea cycle Also, high levels of arginine activate Nacetylglutamate synthase, making more N-acetylglutamate, which activates the cycle Gene expression of urea cycle enzymes increases when needed: – High protein diet – Starvation, when protein is being broken down for energy GENETIC DEFECTS IN THE UREA CYCLE CAN BE LIFE-THREATENING (JC10) § Individuals with genetic defects in any enzyme involved in urea formation cannot tolerate protein-rich diets. § Because amino acids ingested in excess of the daily requirements for protein synthesis are deaminated in liver, producing ammonia that cannot be converted to urea and transported into the bloodstream for excretion (ammonia is highly toxic). § Deficiency of a urea cycle enzyme can produce hyperammonemia and the build-up of one or more urea cycle intermediates. Most of the the urea cycle steps are irreversible, which means that the defect can often be diagnosed by determining which intermediate is present in particularly high concentrations in blood and/or urine. § Although the breakdown of amino acids can have serious health effects in individuals with urea cycle enzyme deficiencies, a protein-free diet is not an option. Humans are incapable of synthesizing nearly half of the amino acids and these must be provided in the diet. Such amino acids are called essential, while other are conditionally essential. JC10: GENETIC DEFECTS IN THE UREA CYCLE POSSIBLE TREATMENTS OF DEFECTS IN THE UREA CYCLE ENZYMES § The administration in the diet of phenylbutyrate can lower the level of ammonia in the blood by promoting urinary excretion of glutamine as phenylacetylglutamine. § The administration in the diet of benzoate (not shown) can lower the level of ammonia in the blood by promoting urinary excretion of glycine as benzoylglycine (hippurate). § Subsequent synthesis of glycine and glutamine to replenish the pool of these intermediates removes ammonia from the bloodstream. MECHANISM OF ACTION OF PHENYLBUTYRATE AND BENZOATE The administration in the diet of benzoate or phenylbutyrate can lower the level of ammonia in the blood by promoting urinary excretion of glycine as benzoylglycine (hippurate) and glutamine as phenylacetylglutamine. Benzoate is initially converted to benzoyl-CoA at the expenditure of ATP. In a second step, benzoyl-CoA reacts with glycine to form benzoylglycine (hippurate) that is then excreted in the urine. The glycine used in this process must be regenerated and ammonia is thus removed. Phenylbutyrate, undergoes a round of β-oxidation, and leads to phenylacetate which is converted to phenylacetyl-CoA at the expense of ATP. Phenylacetyl-CoA reacts with glutamine to form phenylacetylglutamine which can be excreted. Subsequent synthesis of glycine and glutamine to replenish the pool of these intermediates removes ammonia from the bloodstream. Severe hyperammonemia is treated by hemodialysis Other therapies are more specific to a particular enzyme deficiency: - A deficiency in N-acetylglutamate synthetase results in the absence of the normal activator, N-acetylglutamate. This condition can be treated by administering an analogue of N-acetylglutamate, carbamoyl-glutamate (activates carbamoyl phosphate synthetase I). - Arginine is used for example to treat deficiencies of enzymes 1, 2 and 3 of the cycle (can pick up ammonia from CP or Aspartate). Remember to prepare for next lecture: Lehninger’s Biochemistry (8th ed), §chapter 18: p. 639-655