Part III => METABOLISM and ENERGY. 3.5 Protein Catabolism 3.5a Protein Degradation 3.5b Amino Acid Breakdown 3.5c Urea Cycle

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1 Part III => METABOLISM and ENERGY 3.5 Protein Catabolism 3.5a Protein Degradation 3.5b Amino Acid Breakdown 3.5c Urea Cycle

2 Section 3.5a: Protein Degradation

3 Synopsis 3.5a - Dietary proteins are degraded into free amino acids by the collaborative action of digestive proteases - The bulk of free amino acids released from the breakdown of dietary proteins is funneled toward the synthesis of cellular proteins - However, excess amino acids are usually converted to glucose, acetyl-coa, and ketone bodies and thus serve as metabolic fuels - During starvation, the degradation of cellular proteins within tissues also serves as an alternative source of free amino acids that are ultimately broken down into metabolic intermediates for energy production such degradation usually occurs within lysosomes

4 Dietary Protein Degradation Chymotrypsin Ala Ser Phe Ser Lys Gly Ala Arg Trp Thr Asp Tyr Gly Lys Cys Elastase Trypsin - Dietary proteins are degraded into free amino acids by the collaborative action of three major digestive proteases: pepsin, trypsin, chymotrypsin, and elastase - With preference for hydrophobic and aromatic residues, pepsin displays a high degree of promiscuity (or broad specificity) in its ability to cleave peptide bonds

5 Cellular Protein Degradation Degradation Protein Free Amino Acids - Within cells, proteins are constantly turned over ie proteins typically have half-lives ranging from minutes to days (and weeks or more in some cases) regulatory proteins such as transcription factors usually have a high turn over - During starvation, the degradation of cellular proteins within tissues such as liver, kidney, and skeletal muscle serves as an alternative source of free amino acids that are ultimately broken down into metabolic intermediates for energy production - Such degradation usually occurs within lysosomes that harbor selective importers and hydrolytic proteases such as cathepsins for the breakdown of cytosolic proteins into free amino acids in a manner akin to the action of digestive proteases

6 Amino Acid Absorption Intestinal Lumen Brush Border Cell Blood Capillary - The released amino acids from the degradation of cellular and dietary proteins enter the bloodstream, the latter through the digestive tract (small intestine) via a number of amino acid transporters and are subsequently absorbed by other tissues - Once inside the cells, excess dietary amino acids are broken down into metabolic intermediates, many of which enter the Krebs cycle for energy production

7 Exercise 3.5a - Describe how dietary proteins are broken down - Compare the substrate specificities of trypsin and chymotrypsin - What are the major organs where cellular proteins are broken down during times of starvation? - After their release from the proteins, how are free amino acids absorbed into the bloodstream?

8 Section 3.5b: Amino Acid Breakdown

9 Synopsis 3.5b - After their release from dietary/cellular proteins, free amino acids can be broken down into the following metabolites for energy production (or in biosynthetic pathways): - α-ketoglutarate - Succinyl-CoA - Fumarate - Oxaloacetate - Pyruvate - Acetoacetate - Acetyl-CoA - Of these seven metabolites, four are Krebs cycle intermediates: - -ketoglutarate - Succinyl-CoA - Fumarate - Oxaloacetate - Pyruvate and acetoacetate (a ketone body) can be easily converted to acetyl-coa the spark that starts the Krebs cycle ignition by virtue of its ability to donate a two-carbon unit in the form of an acetyl group - In a nutshell, the breakdown products of amino acids essentially serve as a fuel for the Krebs cycle but be aware that acetyl-coa can also be converted into fatty acids!

10 Overview of Amino Acid Breakdown In the context of their catabolic breakdown, amino acids can be divided into two groups: (1) Glucogenic amino acids these are amino acids that can be directly broken down into glucose precursors such as pyruvate, - ketoglutarate, succinyl-coa, fumarate, or oxaloacetate used in the synthesis of glucose (gluconeogenesis) (2) Ketogenic amino acids these are amino acids that can be directly broken down into ketogenic precursors such as acetyl-coa or acetoacetate used in the synthesis of ketone bodies (ketogenesis) Helpful Hints: (a) Of the 20 standard amino acids, only Leu and Lys are NOT glucogenic ie they are exclusively ketogenic! (b) Of the other 18 amino acids, only five amino acids are both glucogenic and ketogenic Trp, Ile, Phe, Thr and Tyr (use WIFTY as a mnemonic!)

11 Products of Amino Acid Breakdown Two major mechanisms involved in amino acid breakdown are: 1) Transamination (cytosolic) 2) Deamination (mitochondrial) pka 9 NH 3 H 2 O <==> NH 4 HO ph = 7.4 => NH 3 = NH 4 Under physiological settings, NH 3 largely exists as NH 4

12 1) Transamination: General Features Aminotransferase - In TRANSAMINATION, the NH 2 group of an amino acid (the donor) is transferred to an - keto acid (the acceptor) the most common -keto acid acceptor is -ketoglutarate - Catalyzed by aminotransferase or transaminase (specific for each amino acid), the transamination reaction of an amino acid with -ketoglutarate produces glutamate and the -keto acid of the original amino acid which is either a simpler metabolite or ultimately converted to one for subsequent oxidation to produce energy - Other than -ketoglutarate, oxaloacetate ( -ketosuccinate) and pyruvate ( -ketopropionate) also serve as important -keto acid acceptors in the context of amino acid transamination - Transamination of amino acids occurs in the cytosol of not only livers cells but also peripheral tissues such as the heart muscle, skeletal muscle, and kidneys

13 1) Transamination: Regenerating -Ketoglutarate (reversible) OOC CH 2 Oxaloacetate Aspartate Aminotransferase OOC CH 2 Aspartate - Glutamate the end-product resulting from the transamination of most amino acids often donates its NH 2 group to oxaloacetate ( -ketosuccinate) to regenerate - ketoglutarate (particularly in liver cells) - Such transamination reaction is catalyzed by aspartate aminotransferase, producing aspartate as a by-product (particularly in liver cells) or the reaction may proceed in the reverse direction producing oxaloacetate (usually enters gluconeogenesis) - In liver cells, aspartate serves as a key metabolite in the urea cycle (next section)

14 1) Transamination: Glutamate to Alanine (reversible) CH 3 Pyruvate Alanine Aminotransferase CH 3 Alanine - In peripheral tissues, glutamate the end-product resulting from the transamination of most amino acids is ultimately converted to alanine (through the alanine cycle) - Catalyzed by alanine aminotransferase, the transfer of NH 2 group of glutamate to pyruvate generates α-ketoglutarate and alanine - Alanine enters the bloodstream and is transported to liver cells alanine thus serves as a nitrogen-carrier between peripheral tissues and liver - In liver cells, the build up of alanine drives the equilibrium in favor of glutamate (the above reaction reverses), which will be ultimately deaminated to NH 3 - Pyruvate meets metabolic fates such as the Krebs cycle or gluconeogenesis

15 1) Transamination: Glutamate to Glutamine (irreversible) O NH 3 Glutamate O C CH 2 CH 2 CH COO Glutamine ATP NH 4 ADP P i Glutamine Synthetase O NH 3 H 2 N C CH 2 CH 2 CH COO - In peripheral tissues, glutamate the end-product resulting from the transamination of most amino acids can also be converted to glutamine through condensation with NH 4 by glutamine synthetase, in what can be envisioned as a pseudo-transamination reaction - Glutamine enters the bloodstream and is transported to liver cells glutamine thus also serves as a nitrogen-carrier between peripheral tissues and liver - In liver cells, glutamine is converted back to glutamate, so that it can be completely deaminated to eliminate NH 4 - Together with alanine, glutamine thus plays a key role in the transport of nitrogen from amino acids in peripheral tissues to liver

16 2) Deamination: General Features H 2 O NH 4 Deaminase - In DEAMINATION, the NH 2 group is removed in the form of NH 4 from an amino acid by a group of enzymes called deaminases the corresponding -keto acid is either a simpler metabolite or ultimately converted to one for subsequent oxidation to produce energy - Deamination of glutamate (and other amino acids) primarily occurs within the mitochondrial matrix of livers cells (and kidney cells to a lesser extent) - Such compartmentalization of deamination within the mitochondrial matrix limits the toxic effect of NH 4 prior to its detoxification via the urea cycle (next section) - After their transport from peripheral tissues into liver cells, the nitrogen-carriers alanine and glutamine are converted back to glutamate, which is subsequently deaminated into -ketoglutarate and NH 4 - In liver cells, alanine undergoes transamination with -ketoglutarate to generate glutamate in a reverse reaction driven by alanine aminotransferase (vide supra) - On the other hand, the liver glutaminase catalyzes direct deamination of the sidechain NH 2 group of glutamine to generate glutamate (vide infra)

17 2) Deamination: Glutamate -Ketoglutarate Glutamate Dehydrogenase - Within liver cells, glutamate the end-product resulting from the transamination of most amino acids is subsequently oxidized by glutamate dehydrogenase to α-ketoglutarate with concomitant release of NH 4 in a reaction termed oxidative deamination - Glutamate dehydrogenase utilizes NAD or NADP as an oxidizing agent - Deamination proceeds by dehydrogenation of the C-N bond followed by hydrolysis of the resulting Schiff base (an imine harboring C=N bond) - -Ketoglutarate meets metabolic fates such as the Krebs cycle - NH 4 enters the urea cycle (next section)

18 2) Deamination: Serine Pyruvate NH 3 Serine HO CH 2 CH COO H 2 O NH 4 Serine Dehydratase Pyruvate O CH 3 C COO - Unlike the transamination of most amino acids into glutamate, serine can be directly deaminated into pyruvate and NH 4 within the mitochondrial matrix of liver cells - Reaction catalyzed by serine dehydratase using H 2 O as a nucleophile to eliminate NH 4 - Pyruvate usually enters Krebs cycle or gluconeogenesis - NH 4 enters the urea cycle (next section)

19 2) Deamination: Threonine -Ketobutyrate OH NH 3 Threonine H 3 C CH CH COO H 2 O NH 4 Threonine Dehydratase -Ketobutyrate O H 3 C CH 2 C COO - Like serine, threonine can be directly deaminated into -ketobutyrate and NH 4 within mitochondrial matrix of liver cells - Reaction catalyzed by threonine dehydratase using H 2 O as a nucleophile to eliminate NH 4 - -Ketobutyrate usually enters Krebs cycle after its conversion into succinyl-coa - NH 4 enters the urea cycle (next section)

20 2) Deamination: Asparagine Aspartate O NH 3 Asparagine Aspartate NH 4 - Asparagine can be partially deaminated into aspartate and NH 4 within mitochondrial matrix of liver cells - Reaction catalyzed by asparaginase using H 2 O as a nucleophile to eliminate NH 4 - Aspartate can undergo transamination to produce oxaloacetate (vide infra), or enter the urea cycle (next section) - NH 4 enters the urea cycle (next section) H 2 N C CH 2 CH COO H 2 O Asparaginase O NH 3 O C CH 2 CH COO

21 2) Deamination: Glutamine Glutamate Glutamine O NH 3 H 2 N C CH 2 CH 2 CH COO Glutamate H 2 O Glutaminase NH 4 O NH 3 - Glutamine can be partially deaminated into glutamate and NH 4 within the mitochondrial matrix of liver cells - Reaction catalyzed by glutaminase - Glutamate can be further deaminated by glutamate dehydrogenase (vide supra) - NH 4 enters the urea cycle (next section) O C CH 2 CH 2 CH COO

22 Exercise 3.5b - Describe the two general metabolic fates of the carbon skeletons of amino acids - List the seven metabolites that represent the end products of amino acid catabolism. Which are glucogenic? Which are ketogenic? - Which amino acids can be broken down into the Krebs cycle intermediates? - Which amino acids can be broken down into pyruvate? - Which amino acids can be broken down into acetyl-coa and/or acetoacetate?

23 Section 3.5c: Urea Cycle

24 Synopsis 3.5c - Excess nitrogen (the NH 2 group) resulting from the breakdown of free amino acids into metabolic fuels is released in the form of NH 3 (strictly NH 4 ) - Where water is plentiful, many aquatic animals directly excrete NH 4 in the urine - In terrestrial vertebrates, NH 4 is converted to less toxic urea primarily in the liver but also in kidneys to a lesser extent via the so-called urea cycle - After its synthesis in the liver, urea is secreted into the bloodstream and ultimately sequestered by the kidneys for excretion in the urine - Reaction of NH 4 (originating from amino acid breakdown) with HCO 3- (eg CO 2 from tissue respiration and decarboxylation) produces urea via the following reaction: NH 3 CO 2 H 2 O aspartate 3ATP H 2 O NH 4 HCO 3 aspartate 3ATP H 2 O urea fumarate 2ADP AMP PP i 2P i Urea (carbamide)

25 Overall Reaction NH 4 H 2 O Urea s two NH 2 groups are derived from NH 4 (ultimately from amino acid breakdown) and aspartate (an amino acid), while the central C=O group hails from the HCO 3- (and H 2 O)

26 The Urea Cycle a largely liver affair! - First ever metabolic cycle discovered by Krebs and Henseleit in It is called cycle rather than a pathway because it cycles ornithine back to itself ie the substrate and the product are identical! - Comprised of five enzymaticallydriven metabolic steps (Steps 1-5) the first two steps occur within the mitochondrial matrix, while latter three in the cytosol of liver cells - Although essential, Steps 1 is technically not an integral component of the urea cycle - Of the four metabolic intermediates of urea cycle, three are non-standard or non-proteinogenic amino acids!

27 Urea Cycle: 1 Carbamoyl Phosphate Synthetase I (CPS-I) O Carbamoyl phosphate CPS NH 4 HCO 3 2ATP H 2 N C OPO 2-3 2ADP P i - In the mitochondrial matrix, NH 4 resulting from the deamination of amino acids is condensed with HCO 3- (eg from tissue respiration and decarboxylation) to generate carbamoyl phosphate cf similarity with urea H 2 N C(O) NH 2 - Reaction is catalyzed by CPS-I and powered by ATP - CPS-I (mitochondrial matrix) is one of the two major forms of CPS CPS-II (cytosolic) uses glutamine as a source of nitrogen to generate carbamoyl phosphate involved in the biosynthesis of pyrimidine nucleotides (see 4.2) - Carbamoyl phosphate is a carboxamide with the formula R C(O)NH 2, the functional group of which is referred to as CARBAMOYL (prefix) or AMIDE (suffix) eg carbamoyl phosphate may also be written as phospoamide! - Carbamoyl phosphate is an activated molecule (cf UDP-glucose in 3.3) in that it can readily donate its carbamoyl moiety C(O)NH 2 to a substrate enter ornithine

28 Urea Cycle: 2 Ornithine Transcarbamoylase (OTC) O NH 3 Carbamoyl phosphate H 2 N C OPO 3 2- Ornithine H 3 N (CH 2 ) 3 CH COO P i OTC O Citrulline NH 3 H 2 N C HN (CH 2 ) 3 CH COO - In the mitochondrial matrix, the carbamoyl moiety C(O)NH 2 of carbamoyl phosphate is transferred to ornithine to generate citrulline both of which are non-standard amino acids in that they play no role in protein biosynthesis - Reaction is catalyzed by OTC producing inorganic phosphate (P i ) as a by-product - Both ornithine (produced in the cytosol) and citrulline (produced in the mitochondrion) require specific transporters located within the inner mitochondrial membrane (IMM) for their transport in and out of mitochondria the next three steps of urea cycle all occur within the cytosol ending with the recycling of ornithine

29 Urea Cycle: 3 Argininosuccinate Synthetase (ASS) Aspartate COO O Citrulline NH 3 OOC CH 2 CH NH 3 H 2 N C HN (CH 2 ) 3 CH COO ATP ASS AMP PP i COO NH 2 NH 3 Argininosuccinate OOC CH 2 CH NH C HN (CH 2 ) 3 CH COO - In the cytosol, aspartate is condensed with citrulline to generate argininosuccinate the third non-standard amino acid in the urea cycle - Reaction is catalyzed by ASS in the presence of ATP, producing AMP and pyrophosphate (PP i ) as by-products the spontaneous hydrolysis of the latter drives the forward reaction - Of the two NH 2 groups in urea H 2 N C(O) NH 2, one is supplied by NH 4 (resulting from the deamination of glutamate) and the other by aspartate

30 Urea Cycle: 4 Argininosuccinase (ASL) COO NH 2 NH 3 Argininosuccinate OOC CH 2 CH NH C HN (CH 2 ) 3 CH COO ASL Fumarate COO NH 2 NH 3 Arginine OOC HC CH H 2 N C HN (CH 2 ) 3 CH COO - In the cytosol, argininosuccinate is cleaved (or split up) into fumarate and arginine - Reaction is catalyzed by ASL (argininosuccinase or argininosuccinate lyase) recall that lyase breaks chemical bonds by means other than hydrolysis - Fumarate is ultimately converted to oxaloacetate by cytosolic enzymes in a manner akin to its fate in the Krebs cycle (see 3.6) oxaloacetate usually enters gluconeogenesis - Arginine continues to travel along the urea cycle as it serves as the precursor of urea

31 Urea Cycle: 5 Arginase (ARG) NH 2 NH 3 Urea H 2 N C HN (CH 2 ) 3 CH COO O H 2 O H 2 N C NH 2 Arginine ARG Ornithine NH 3 H 3 N (CH 2 ) 3 CH COO - In the cytosol, arginine is hydrolyzed into ornithine and urea - Reaction is catalyzed by ARG using H 2 O as a nucleophile to eliminate urea ie arginase is an hydrolase! - Ornithine is shuttled back into the mitochondrial matrix for another round of detoxification of NH 4 - Urea is secreted into the bloodstream and ultimately sequestered by the kidneys for excretion in the urine

32 Exercise 3.5c - What is the difference between carbamoyl and amide functional groups? - Compare the chemical structures of carbamoyl phosphate and urea (carbamoyl amine) - Summarize various steps of the urea cycle