1. III. METABOLIC BIOCHEMISTRY
§3.4 Protein Catabolism
§3.4a Protein Degradation
§3.4b Amino Acid Breakdown
§3.4c Urea Cycle

2. §3.4a Protein Degradation

3. Synopsis 3.4a
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
—Ala—Ser—Phe—Ser—Lys—Gly—Ala—Arg—Trp—Thr—Asp—Tyr—Gly—Lys—Cys—
Trypsin
Chymotrypsin
Elastase
In addition to pepsin, dietary proteins are degraded into free amino acids by the collaborative action of three other major digestive proteases: trypsin, chymotrypsin, and elastase
With preference for hydrophobic and aromatic residues, pepsin displays a rather high degree of promiscuity (or broad specificity) in its ability to cleave peptide bonds

5. Cellular Protein Degradation
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 3.4a 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 dietary proteins, how are free amino acids absorbed into the bloodstream?

8. §3.4b Amino Acid Breakdown

9. Synopsis 3.4b
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. Glucogenic and Ketogenic Precursors
Amino Acid Breakdown:
Glucogenic and Ketogenic Precursors
In the context of their catabolic breakdown, amino acids can be divided into two groups:
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)
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:
Of the 20 standard amino acids, only Leu and Lys are NOT glucogenic—ie they are exclusively ketogenic!
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. NH3 + H2O <==> NH4+ + HO-
Amino Acid Breakdown:
Transamination and Deamination
Two major mechanisms involved in amino acid breakdown are:
1) Transamination (cytosolic)
2) Deamination (mitochondrial)
NH3 + H2O <==> NH4+ + HO-
pKa  9
@ pH = 7.4 => NH3 = NH4+
Under physiological settings, NH3 largely exists as NH4+

12. Relationship to Mono- and Dicarboxylic Acids
Amino Acid Breakdown:
Relationship to Mono- and Dicarboxylic Acids
Monocarboxylate Anions
4:1
Butyrate
(Butanoate) -
5:1
Valerate
(Pentanoate) -
1:1
Formate
(Methanoate) -
2:1
Acetate
(Ethanoate) -
3:1
Propionate
(Propanoate) -
Dicarboxylate Dianions
4:2
Succinate
(Butanedioate) -
2:2
Oxalate
(Ethanedioate) -
3:2
Malonate
(Propanedioate) -
5:2
Glutarate
(Pentanedioate) -

13. 1) Transamination: General Features
(-Aminoglutarate)
Aminotransferase
In transamination, the —NH2 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

14. 1) Transamination: Regenerating -Ketoglutarate (reversible)
Aspartate
CH2
OOC —
Aminotransferase
Oxaloacetate
(-Aminoglutarate)
(-Aminosuccinate)
(-Ketosuccinate)
Glutamate—the end-product resulting from the transamination of most amino acids—often donates its —NH2 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)

15. 1) Transamination: Glutamate to Alanine (reversible)
Aminotransferase
CH3
Pyruvate
(-Aminoglutarate)
(-Ketopropionate)
(-Aminopropionate)
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 –NH2 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 NH3
Pyruvate meets metabolic fates such as the Krebs cycle or gluconeogenesis

16. 1) Transamination: Glutamate to Glutamine (irreversible)
Synthetase
ATP + NH4+
ADP + Pi
O NH3
H2N—C—CH2—CH2—CH—COO +
Glutamate
O—C—CH2—CH2—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 NH4+ 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 partially deaminated to glutamate—which is further deaminated to -ketoglutarate to completely eliminate NH4+ (vide infra)
Together with alanine, glutamine thus plays a key role in the transport of nitrogen from amino acids in peripheral tissues to liver

17. 2) Deamination: General Features
Deaminase
H2O
NH4+
In deamination, the —NH2 group is removed in the form of NH4+ 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 most other amino acids) primarily occurs within the mitochondrial matrix of liver cells (and kidney cells to a lesser extent)
Such compartmentalization of deamination within the mitochondrial matrix limits the toxic effect of NH4+—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 NH4+
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 — NH2 group of glutamine to generate glutamate (vide infra)

18. 2) Deamination: Glutamine  Glutamate (irreversible)
Glutaminase
NH4+
O NH3
H2N—C—CH2—CH2—CH—COO +
Glutamine
H2O
Glutamate
O—C—CH2—CH2—CH—COO
Glutamine—largely from peripheral tissues as a nitrogen-carrier—can be partially deaminated into glutamate and NH4+ within the mitochondrial matrix of liver cells
Reaction catalyzed by glutaminase— using H2O as a nucleophile to eliminate NH4+
Glutamate can be further deaminated by glutamate dehydrogenase (vide supra)
NH4+ enters the urea cycle (next section)

19. Glutamate Dehydrogenase
2) Deamination: Glutamate  -Ketoglutarate (irreversible)
Glutamate Dehydrogenase
(-Aminoglutarate)
Within the mitochondrial matrix of 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 NH4+ 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
NH4+ enters the urea cycle (next section)

20. 2) Deamination: Asparagine  Aspartate (irreversible)
Asparaginase
NH4+
O NH3
H2N—C—CH2—CH—COO +
Asparagine
H2O
Aspartate
O—C—CH2—CH—COO
Asparagine can be partially deaminated into aspartate and NH4+ within mitochondrial matrix of liver cells
Reaction catalyzed by asparaginase—using H2O as a nucleophile to eliminate NH4+
Aspartate can undergo transamination to produce oxaloacetate (vide supra), or enter the urea cycle (next section)
NH4+ enters the urea cycle (next section)

21. 2) Deamination: Threonine  -Ketobutyrate (irreversible) + OH NH3
Dehydratase
NH4+
OH NH3
H3C—CH—CH—COO
-Ketobutyrate
H2O O
H3C—CH2—C—COO
Like serine, threonine can be directly deaminated into -ketobutyrate and NH4+ within the mitochondrial matrix of liver cells
Reaction catalyzed by threonine dehydratase—using H2O as a nucleophile to eliminate NH4+
-Ketobutyrate usually enters Krebs cycle after its conversion into succinyl-CoA
NH4+ enters the urea cycle (next section)

22. 2) Deamination: Serine  Pyruvate (irreversible)
Dehydratase
NH4+
NH3
HO—CH2—CH—COO +
Pyruvate
H2O O
CH3—C—COO
Unlike the transamination of most amino acids into glutamate, serine (as well as alanine and cysteine) can be directly deaminated into pyruvate and NH4+—these reactions largely occur within the cytosol of liver cells (possibly due to a major role of pyruvate in gluconeogenesis)
Serine deamination is catalyzed by serine dehydratase—using H2O as a nucleophile to eliminate NH4+
Pyruvate usually enters Krebs cycle or gluconeogenesis
NH4+ enters the urea cycle (next section)

23. Exercise 3.4b
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?

24. §3.4c Urea Cycle

25. Synopsis 3.4c
Excess nitrogen (the –NH2 group) resulting from the breakdown of free amino acids into metabolic fuels is released in the form of NH3 (strictly NH4+)
Where water is plentiful, many aquatic animals directly excrete NH4+ in the urine
In terrestrial vertebrates, NH4+ 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 NH4+ (NH3 originating from amino acid breakdown) with HCO3- (eg CO2 from tissue respiration and decarboxylation) produces urea via the following reaction:
NH3 + CO2 + H2O + aspartate + 3ATP + H2O
NH4+ + HCO3 + aspartate + 3ATP + H2O
urea + fumarate + 2ADP + AMP + PPi + 2Pi
Urea
(carbamide)

26. Overall Reaction NH4+ H2O
Urea’s two –NH2 groups are derived from NH4+ (ultimately from amino acid breakdown) and aspartate (an amino acid), while the central C=O group hails from the HCO3-

27. The Urea Cycle—a largely liver affair!
First ever metabolic cycle discovered by Krebs and Henseleit in 1932
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 enzymatically-driven 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!

28. NH4+ + HCO3 + 2ATP H2N—C—OPO32- + 2ADP + Pi
Urea Cycle: 1 Carbamoyl Phosphate Synthetase I (CPS-I) O
NH HCO3 + 2ATP H2N—C—OPO ADP + Pi
CPS
Carbamoyl
phosphate
In the mitochondrial matrix, NH4+ resulting from the deamination of amino acids is condensed with HCO3- (eg from tissue respiration and decarboxylation) to generate carbamoyl phosphate—cf similarity with urea H2N—C(O)—NH2
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 §4.1)
Carbamoyl phosphate is a carboxamide with the formula R—C(O)NH2, 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 §3.2) in that it can readily donate its carbamoyl moiety —C(O)NH2 to a substrate–enter ornithine

29. Urea Cycle: 2 Ornithine Transcarbamoylase (OTC) + O NH3
H2N—C—OPO H3N—(CH2)3—CH—COO
OTC
O NH3
H2N—C—HN—(CH2)3—CH—COO Pi
Carbamoyl
phosphate
Ornithine
Citrulline +
In the mitochondrial matrix, the carbamoyl moiety —C(O)NH2 of carbamoyl phosphate is transferred to ornithine (cf lysine) to generate citrulline (carbamoyl ornithine!)—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 (Pi) 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

30. OOC—CH2—CH—NH—C—HN—(CH2)3—CH—COO
Urea Cycle: 3 Argininosuccinate Synthetase (ASS)
COO
OOC—CH2—CH—NH3
ASS
O NH3
H2N—C—HN—(CH2)3—CH—COO
AMP + PPi
Aspartate
Citrulline +
COO NH NH3
OOC—CH2—CH—NH—C—HN—(CH2)3—CH—COO
Argininosuccinate
ATP
In the cytosol, aspartate is condensed with citrulline to generate argininosuccinate (succinylarginine!)—the third non-standard amino acid in the urea cycle
Reaction is catalyzed by ASS in the presence of ATP, producing AMP and pyrophosphate (PPi) as by-products—the spontaneous hydrolysis of the latter drives the forward reaction
Of the two —NH2 groups in urea H2N—C(O)—NH2, one is supplied by NH4+ (resulting from the deamination of glutamate) and the other by aspartate

31. H2N—C—HN—(CH2)3—CH—COO OOC—CH2—CH—NH—C—HN—(CH2)3—CH—COO
Urea Cycle: 4 Argininosuccinase (ASL)
COO
OOC—HC—CH
ASL
NH NH3
H2N—C—HN—(CH2)3—CH—COO
Fumarate +
COO NH NH3
OOC—CH2—CH—NH—C—HN—(CH2)3—CH—COO
Argininosuccinate
Arginine
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 §3.5)—oxaloacetate usually enters gluconeogenesis
Arginine continues to travel along the urea cycle as it serves as the precursor of urea

32. H2N—C—HN—(CH2)3—CH—COO
Urea Cycle: 5 Arginase (ARG)
ARG
NH3
H3N—(CH2)3—CH—COO +
H2O
Ornithine
NH NH3
H2N—C—HN—(CH2)3—CH—COO
Arginine O
H2N—C—NH2
Urea
In the cytosol, arginine is hydrolyzed into ornithine and urea
Reaction is catalyzed by ARG using H2O 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 NH4+
Urea is secreted into the bloodstream and ultimately sequestered by the kidneys for excretion in the urine

33. Exercise 3.4c
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