Protein digestion begins with pepsin and HCL in the stomach breaking proteins into smaller peptides. In the small intestine, pancreatic proteases including trypsin further break peptides into amino acids. Amino acids are actively absorbed into intestinal cells via sodium-coupled amino acid transporters and released into the bloodstream. Excess amino acid nitrogen from dietary and cellular proteins is converted to ammonia in tissues then transported to the liver. In the liver, ammonia is incorporated into urea through the urea cycle and excreted in urine.

1. 8. PROTEIN
METABOLISM

2. CONTENTS
• Protein Metabolism
• 1. Digestion and absorption of Proteins
• 2. Essential and non-essential amino acids
• 3. Catabolism of amino acid nitrogen
• a. Transaminations
• b. Oxidative deamination
c. Biosynthesis of urea
d. Clinical correlates
 Liver diseases and transaminases
 Liver diseases and hyperammonia
 Kidney diseases and BUN
4. Catabolism of amino acid carbon skeleton
 5. Amino acids as a source of special peptide
a. creatine phosphate
b. Glutathione

3. Digestion& absorptionof Proteins
DIGESTION OF PROTEINS
Proteolytic enzymes (also called proteases) break down dietary
proteins into their constituent amino acids in the stomach and the
intestine. Many of these digestive proteases are synthesized as larger,
inactive forms known as zymogens. After zymogens are secreted into
the digestive tract, they are cleaved to produce the active proteases.
• Proteins are digested by proteases which hydrolyze specific peptide
bonds. Proteases belong to two types:
Exopeptidases such as Carboxypeptidases and Aminopeptidases
hydrolyze only a terminal peptide bond holding the last amino acid
residue of the peptide chain, and release that end amino acid.
Endopeptidases such as pepsin and trypsin hydrolyze specific peptide
bonds even deep inside the peptide chain to cleave it into smaller
peptides.

4. Proteases are classified according to such specific critical
groups or residues at their active sites as are essential for
their activity:
• Serine proteases such as trypsin and chymotrypsin possess a
critical serine residue at the active site.
• Thiol proteases such as papain possess at the actives site a
critical cysteine residue whose side chain SH must remain
free for their activity.
• Zinc proteases such as carboxypeptidases A and B require at
the active site a tight bound Zn2+ for activity.
• Acid proteases such as pepsin possess at the active site a
critical dicarboxylic amino acid residue with a side chain
COOH and have acidic optimum pHs.

5. Digestion in Stomach
Protein digestion is started by pepsin-HCL in stomach.
a)Action of pepsin and HCL:
• Pepsin occurs in the gastric juice. It is secreted in the gastric juice
as an inactive proenzyme, pepsinogen. The latter undergoes
irreversible covalent activation to pepsin in the gastric lumen
either at a pH below 2 provided by gastric HCL, or by
autocatalysis by already active pepsin at pH around 4.6.
• Gastric HCL helps in digestion because it lowers the gastric pH
below 2 to activate pepsinogen to pepsin and maintain the
gastric pH 1.6-3.2 for optimal pepsin activity.
• Pepsin is an endopeptidase. It hydrolyzes peptide bonds even
deep inside the peptide chain to digest native and denatured
proteins to proteoses and peptones.
• Action of pepsin is terminated in the duodenum due to its
inactivation by trypsin and alkalinities of pancreatic juice and
bile.

6. Digestion by pancreatic juice
• Pancreatic juice contains five proteases- trypsin,
chymotrypsin, elastase and carboxypeptidases A and B, all
secreted as inactive proenzymes: of trypsin, activated by
intestinal enteropeptidase, activates the other proenzymes
and the remaining trypsinogen molecules. Thus, the
activation of all the pancreatic proteases is triggered by
enteropeptidase.
• Pancreatic tissue is effectively protected against its own
proteases because they are all secreted as inactive
proenzymes.
a) Action of trypsin:
• Inactive trypsinogen, secreted in the intestinal lumen, is
hydrolyzed to active trypsin by enteropeptidase of intestinal
juice at pH 5.2-6.0. But with the rise in pH to 7.9, already
activated trypsin hydrolyze more trypsinogen molecules to
trypsin.

7. b) Action of chymotrypsin:
• Chymotrypsin is secreted as inactive chymotrypsinogen. In the
intestinal lumen, trypsin hydrolyzes the peptide bond between
the alpha-COOH of arginine and the alpha-NH2 of isoleucine in
chymotrypsinogen to release a 15 residue inactive peptide from
the N-terminal end of the latter. This changes chymotrypsinogen
to active chymotrypsin. They digest protein and peptides to
smaller peptides by hydrolyzing peptide bonds connected with
the alpha-COOH of either aromatic amino acids or amino acids
with large nonpolar sidechains.
c) Action of elastase:
• Inactive proelastase is hydrolyzed by trypsin into the active
enzyme and an inactive peptide. Elastase is a serine protease and
endopeptidase. It hydrolyzes specifically the peptide bonds
connected to the alpha-COOH of neutral aliphatic amino acids
with small uncharged sidechains. It digests proteins, particularly
elastins to peptides.

8. d) Action of carboxypeptidases:
• Both carboxypeptidases are exopeptidases,
secreted as inactive procarboxypeptidases and
activated by trypsin. Both are zinc proteases, with
pH optima at 7.5-8, and ineffective in hydrolyzing
dipeptides. Trypsin activates carboxypeptidases A.
The latter hydrolyzes the C-terminal peptide bond
holding either an aromatic amino acid or branched
chain aliphatic amino acid. Carboxypeptidase B,
formed from inactive procarboxypeptidase B by
trypsin, hydrolyzes the C-terminal peptide bond
holding a basic amino acid as the end amino acid.

10. Digestion by Intestinal juice
• Intestinal proteases include enteropeptidase,
aminopeptidases and dipeptidases. Enteropeptidase and
aminopeptidase are glycoproteins which mostly remain
anchored to the microvillus membrane of enterocytes and
project into the intestinal lumen. But most dipeptidases
occur in the enterocyte cytoplasm. Except enteropeptidase,
others are exopeptidases hydrolyzing only terminal peptide
bonds of small peptides produced by earlier digestion.
A) Action of enteropeptidase:
• Enteropeptidase is an endopeptidase. In the intestinal
lumen, it hydrolyzes trypsinogen into active trypsin and an
inactive hexapeptide. It can also digest other proteins into
smaller peptides by hydrolyzing peptide bonds connected
with the COOH group of lysine

11. B) Action of aminopeptidases:
• These enzymes of the luminal membrane hydrolyze the N-
terminal peptide bond of a peptide chain to release the N-
terminal amino acid. Successive actions of aminopeptidases
change the peptide step-wise into a dipeptide, but cannot
dipeptides.
C) Action of dipeptidases:
• Dipeptidases such as glycylglycine dipeptidase hydrolyze
dipeptides, each into two molecules of amino acids, inside the
enterocytes to complete the digestion of proteins.

12. Absorption of proteins
Digestion products of proteins are absorbed mainly as amino acids
and to smaller extend as small oligopeptides from jejunum.
Oligopeptides are hydrolyzed into amino acids by peptidases of
the microvillus membrane during their absorption. Following
evidences indicate that L-amino acids, in contrast to D-amino acids
absorbed by simple diffusion, are absorbed by carrier-mediated
processes, mostly coupled with Na+-K+ pump.
a) The rate of absorption of L-amino acid far exceeds that of its D
isomer and is independent of the diffusion coefficient and
concentration gradient.
b) L-amino acid absorption is depressed by cold, hypoxia, metabolic
inhibitors like dinitrophenol and cyanide and sodium pump
inhibitors like ouabain.

13. Processes:
• L-amino acids are absorbed actively by the coupled
activities of the Na+-K+ pump or Na+-K+ ATPase of
the basolateral membrane of the enterocytes and a
number of Na+-amino acid cotransporters of the
microvillus membrane. The Na+-K+ ATPase extrudes
three Na+ ions from the enterocyte to the
interstitial fluid in exchange of two K+ ions brought
into the cell, both across its basolateral membrane,
at the cost of a high-energy bond of ATP. This
maintains a lower Na+ concentration in the cell
than that in the intestinal lumen.

14. On the luminal surface of the microvillus membrane, Na+ and a
specific amino acid from the intestinal contents bind
successively to separate binding sites of a specific Na+ -amino
acid cotransporter which then transports both across the
luminal membrane into the cell, guided by the inward Na+
concentration gradient, and releases both Na+ and the amino
acid in to the cytoplasm to maintain high intracellular
concentrations of both.
Different Na+-amino acid co-transporters bind to and transfer in
this way amino acids of specific classes across the luminal
membrane; example, a carrier for nonpolar amino acids such as
phenylalanine and methionine, another carrier for the imino
acids, proline and hydroxy proline, and still another for neutral
amino acids, pyridoxal phosphate appears to participate in the
active absorption of amino acids.

15. Absorption of unhydrolyzed proteins and
polypeptides may lead to antigenic reactions like food
allergies. In nontropical sprue, some defect in the
intestinal mucosa may cause the enterocytes to
absorb unhydrolyzed hexa and hepta peptides
produced by gastric and pancreatic digestions of the
wheat protein, gluten. These absorbed oligopeptides
may produce intestinal lesions and may also
stimulate antibody production by their antigenic
effects.

17. Essentialand Non-essentialAmino Acids

19. 2. Catabolism of Proteins & of Amino Acid
Nitrogen- Urea cycle
In human tissues, alpha-NH2 group of amino acids, derived
either from the diet or breakdown of tissue proteins,
ultimately is converted first to NH3 and then to urea and is
excreted in the urine.
The formation of NH3 and urea can be discussed under the
following heads:
• Transamination
• Deamination
- Oxidative deamination
• Formation of urea

21. A. Transamination

26. B. Glutamate dehydrogenase: the oxidative deamination of amino
acids:
In contrast to transamination reactions that transfer amino groups,
oxidative deamination by glutamate dehydrogenase results in the
liberation of the amino group as free ammonia (NH3).
They provide α-keto acids that can
enter the central pathway of
energy metabolism, and ammonia,
which is a source of nitrogen in
urea synthesis.

28. C. Transport of ammonia to the liver:
Two mechanisms are available in humans for the transport of
ammonia from the peripheral tissues to the liver for its ultimate
conversion to urea.
The first, found in most tissues, uses glutamine synthetase to
combine ammonia (NH3) with glutamate to form glutamine—a
nontoxic transport form of ammonia.
The glutamine is transported in the blood to the liver where it is
cleaved by glutaminase to produce glutamate and free ammonia.
The second transport mechanism, used primarily by muscle,
involves transamination of pyruvate (the end product of aerobic
glycolysis) to form alanine. Alanine is transported by the blood to
the liver, where it is converted to pyruvate, again by
transamination.
In the liver, the pathway of gluconeogenesis can use the pyruvate
to synthesize glucose, which can enter the blood and be used by
muscle—a pathway called the glucose-alanine cycle.

30. UREA CYCLE
Urea is the major disposal form of amino groups
derived from amino acids, and accounts for about
90% of the nitrogen-containing components of
urine.
The carbon and oxygen of urea are derived from
CO2. Urea is produced by the liver, and then is
transported in the blood to the kidneys for
excretion in the urine.

32. Liver diseases and Transaminases

34. contacts

35. Liverdiseasesand hyperammonaemia
Clinical aspects of Ammonia
• In addition to NH3 formed in the tissues, a considerable
quantity of NH3 is produced in the gut by intestinal
bacterial flora, both from dietary proteins and from
urea present in fluids secreted in the GI tract.
• This NH3 is absorbed from the intestine into portal
venous blood which contains relatively high
concentration of NH3 as compared to systemic blood.
• Under normal conditions of health, liver promptly
removes the NH3 from the portal blood, so that blood
leaving the liver is virtually NH3- free. This is essential
since even small quantities of NH3 are toxic to CNS.
• In man, normal blood level of NH3 varies from 40-70
microgram/100ml.

36. Hyperammonaemia: Hyperammonaemia is associated
with comatose state such as may occur in hepatic failure.
May be of 2 types:-
1. Acquired hyperammonaemia: is usually the result of
cirrhosis of the liver with the development of a collateral
circulation, which shunts the portal blood around the organ,
there by severely reducing the synthesis of urea.
2. Inherited hyperammonaemia: results from genetic defects
in the urea cycle enzymes.
The symptoms of NH3 intoxication include:
• A peculiar flapping tremor
• Slurring of speech
• Blurring of vision
• And in severe cases follows to coma and death

37. Kidney diseases and BUN
Clinical Significance of Urea
• A moderately active man consuming about 300gm
carbohydrates, 100gm of fats and 100gm of proteins daily must
excrete about 16.5gm of N daily. 95% is eliminated by the
kidneys and the remaining 5% for the most part as N in the
faeces.
• Normal level: The concentration of urea in normal blood plasma
from a healthy fasting adult ranges from 20-40mg %.
• Increase of levels: Increase in blood urea may occur in a number
of diseases in addition to those in which the kidneys are
primarily involved. The causes can classified as:
• Prerenal
• Renal
• Postrenal

38. Pre-renal: Most important are conditions in which
plasma volume/body-fluids are reduced:
• Salt and water depletion
• Severe and protracted vomiting as in pyloric and
intestinal obstruction
• Severe and prolonged vomiting
• Pyloric stenosis with severe vomiting
• Haematemesis
• Haemorrhage and shock; shock due to severe
burns
• Ulcerative colitis with severe chloride loss

39. Renal: The blood urea can be increased in all forms of
kidney diseases:
• In acute glomerulonephritis
• In early stages of Type-II nephritis the blood urea
may not be increased, but in later stages with renal
failure, blood urea rises
• Other conditions are malignant nephrosclerosis,
chronic pyelonephritis and mercurial poisoning
• In diseases such as hydronephrosis, renal
tuberculosis; small increase are seen but depends
on extent of kidney damage

40. • Post-renal diseases: These lead to increase in
blood urea, when there is obstruction to urine
flow. This causes retention of urine and so
reduces the effective filtration pressure at the
glomeruli; when prolonged, produces
irreversible kidney damage.

41. BUN
• Serum urea is sometimes expressed in terms of its
nitrogen, because nitrogenous substances were
analyzed by Kjeldahl method. Such expression of
Urea-N or blood urea nitrogen (BUN) is very
common.
• Molecular weight of urea is 60 and each gram mol of
urea contains 28 gram of nitrogen. Thus a serum
concentration of 28 mg/dL of BUN is equivalent to
60 mg/dL of urea.

42. o 3. Catabolism of the carbon skeletons of
amino acids
• The pathways by which amino acids are catabolized are
conveniently organized according to which one (or more) of the
following seven intermediates listed below is produced from a
particular amino acid.
A. Amino acids that form oxaloacetate
B. Amino acids that form α-ketoglutarate via glutamate
C. Amino acids that form pyruvate
D. Amino acids that form fumarate
E. Amino acids that form succinyl CoA: methionine
F. Other amino acids that form succinyl CoA
G. Amino acids that form acetyl CoA or acetoacetyl CoA

43. A. Amino acids that form oxaloacetate
Asparagine is hydrolyzed by asparaginase, liberating ammonia and
aspartate. Aspartate loses its amino group by transamination to
form oxaloacetate.

44. B. Amino acids that form α-ketoglutarate via
glutamate.
1. Glutamine: This amino acid is converted to glutamate and
ammonia by the enzyme glutaminase. Glutamate is converted to α-
keto glutarate by transamination, or through oxidative deamination
by glutamate dehydrogenase.

45. 2. Proline: This amino acid is oxidized to glutamate. Glutamate is
transaminated or oxidatively deaminated to form α-ketoglutarate.
3. Arginine: This amino acid is cleaved by arginase to produce
ornithine. [Note: This reaction occurs primarily in the liver as part of
the urea cycle.]
• 4. Histidine: This amino acid is oxidatively deaminated by histidase
to urocanic acid, which subsequently forms N-formimino glutamate
(FIGlu). FIGlu donates its formimino group to tetra-hydro folate
(THF), leaving glutamate, which is degraded as described above.

46. C. Amino acids that form pyruvate
1. Alanine: This amino acid loses its amino group by reversible
transamination to form pyruvate. [Note: Alanine is the major
gluconeogenic amino acid.]
2. Serine: This amino acid can be converted to glycine and
N5,N10-methylenetetrahydrofolate. Serine can also be
converted to pyruvate by serine dehydratase

47. 3. Glycine: This amino acid can be converted to serine by the
reversible addition of a methylene group from N5,N10-
methylene -tetrahydrofolic acid or oxidized to CO2 and NH3.

48. 4. Cystine: This amino acid is reduced to cysteine, using NADH + H+ as
a reductant. Cysteine undergoes desulfuration to yield pyruvate.

49. 5. Threonine: This amino acid is converted to pyruvate or to α-
ketobutyrate, which forms succinyl CoA.
D. Amino acids that form fumarate
1. Phenylalanine and tyrosine: Hydroxylation of phenylalanine
produces tyrosine. This reaction, catalyzed by tetra -hydrobiopterin-
requiring phenylalanine hydroxylase, initiates the catabolism of
phenylalanine. Thus, the metabolism of phenylalanine and tyrosine
merge, leading ultimately to the formation of fumarate and
acetoacetate.

50. E. Amino acids that form succinyl CoA: methionine
Methionine is one of four amino acids that form succinyl CoA. This
sulfur-containing amino acid is converted to S-adenosyl methionine
(SAM). Methionine is also the source of homocysteine—a metabolite
associated with atherosclerotic vascular disease.

52. F. Other amino acids that form succinyl CoA
Degradation of valine, isoleucine, and threonine also results in the
production of succinyl CoA—a tricarboxylic acid (TCA) cycle
intermediate and glucogenic compound.
1. Valine and isoleucine: These amino acids are branched-chain
amino acids that generate propionyl CoA, which is converted to
succinyl CoA by biotin- and vitamin B12–requiring reactions.

55. 2. Threonine: This amino acid is dehydrated to α-ketobutyrate,
which is converted to propionyl CoA and then to succinyl CoA.
Threonine can also be converted to pyruvate.

56. G. Amino acids that form acetyl CoA or acetoacetyl CoA
• Leucine, isoleucine, lysine, and tryptophan form acetyl CoA
or aceto acetyl CoA directly, without pyruvate serving as an
intermediate.

57. Amino acids as a source of special peptides
• Amino acids are precursors of many nitrogen-
containing compounds including porphyrins, which, in
combination with ferrous (Fe2+) iron, form heme.
Other important N-containing compounds derived from
amino acids include the catechol - amines (dopamine,
norepinephrine, and epinephrine), creatine, histamine,
serotonin, melanin and glutathione.
• Creatine and glutathione are two nitrogenous
compounds which are connected with protein
metabolism.

58. 1. Creatine
• It is a normal constituent of the body. It is present in
muscle, brain, liver, testis and in blood. It can occur in
free form and also as phosphorylated form. The
phosphorylated form is called as creatine phosphate.
Total amount in adult human body is approximately
120gm. 98% of the total amount is present in muscles,
of which 80% occurs in phosphorylated form.
• Urinary excretion in normal health is in the form of
creatinine and it is only 2% of the total. In males it is
1.5-2.0gm in 24 hr urine, and in females, it varies from
0.8-1.5gm.

59. Biosynthesis of creatine
Synthesis: Creatine is synthesized from glycine and the
guanidine group of arginine, plus a methyl group from
SAM (see Figure). Creatine is reversibly phosphorylated
to creatine phosphate by creatine kinase, using ATP as
the phosphate donor.

61. • During muscle contraction, creatine phosphokinase (CPK)
transfers the high-energy phosphate of creatine
phosphate to ADP to regenerate ATP. Creatine phosphate
is spontaneously changed in muscles to Pi and creatinine.
The latter is excreted in the urine.

62. 2. Glutathione
• Glutathione is a tri-peptide of three amino acids,
glutamic acid, cysteine and glycine.
• It is an important reducing agent in the tissues.
• Oxidised glutathione G-S-S-G is harmful to the tissues,
specially to RB cells and is converted to reduced
glutathione G-SH, which is required for the integrity of
RB cells membrane.