Protein metabolism

1
Protein metabolism

• BY
• Dr. NAGLAA IBRAHIM AZAB
• Lecturer Of Medical Biochemistry Molecular
  Biology
• BENHA FACULTY OF MEDICINE

2
PROTEIN STRUCURE
3
Protein structure

• a- a.a ---- a- a.a ---- a- a.a ---- a- a.a
  ---------- a- a.a
• 1 2 3
  4 n
• 
  Peptide linkages
• a- amino acid
• RCHCOOH
• 
  NH2
• R1 R2
  Rn
• NH2 -- CHCO---- NH-- CHCO----------CO---- NH-
  CHCOOH
• Amino terminus peptide
  linkages carboxyl terminus

4
Classification of amino acids

• Structural classification according to
  the chemical structure of the side chain
  (R)
• Nutritional classification
  (essensial non essential a.as)
• Metabolic classification according to
  the fate of amino acids inside the body
  (glucogenic,ketogenic and mixed a.as)

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(No Transcript)
6
Aliphatic a.as
Neutral a.as Contain One NH2 one
COOH
Amidic a.as Contain One NH2 one
COOH One NH2CO(amidic group)
Acidic a.as Contain One NH2 two
COOH
Basic a.as Contain gtOne NH2 one
COOH
7
Aliphatic neutral a.as

• WITH HYDROCARBON SIDE CHAIN
  ---------------------------------
  -----------------
• 1-Glycine(2C) CH2COOH
  
  
  
• 
  
• 2-Alanine(3C)
  CH3CHCOOH
• 3-Valine(5C)
  CH3CHCHCOOH
• 4-Leucine(6C)
  CH3CHCH2CHCOOH
• 5-Isoleucine(6C)
  CH3CH2CHCHCOOH

I NH2
I NH2
I I CH3NH2
I NH2
I CH3
I NH2
I CH3
8

• WITH HYDROXYL (OH ) CONTAINING SIDE CHAIN
• --------------------------------------------------
  ---------------------
• 1-Serine(3C)
  CH2CHCOOH
• 2-Homoserine(4C) CH2CH2CHCOOH
• 3-Threonine(4C) CH3CHCHCOOH

I OH
I OH
I NH2
I OH
I NH2
I OH
I NH2
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WITH SULFUR (S) CONTAINING SIDE
CHAIN--------------------------------------------
-------------------

• 1-Cysteine(3C)
  CH2CHCOOH
• 2-Homocysteine(4C) CH2CH2CHCOOH
• 3-Methionine(4C)
  CH2CH2CHCOOH
• 4-Cystine
  CH2CHCOOH
• 
  S
• 
  S
• 
  CH2CHCOOH

I SH
I NH2
I SH
I NH2
I S-CH3
I NH2
I NH2
I NH2
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• Aliphatic
• Amidic a.as
• Aspargine (4C)
• NH2OCCH2CHCOOH
• Glutamine (5C)
• NH2OCCH2CH2CHCOOH

• Aliphatic
• acidic a.as
• Aspartic acid (4C)
• HOOCCH2CHCOOH
• Glutamic acid (5C)
• HOOCCH2CH2CHCOOH

I NH2
I NH2
I NH2
I NH2
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Aliphatic Basic a.as

• 1-Lysine (6C) CH2CH2CH2CH2CHCOO
  H
• 2-Arginine (5C1)
  CH2CH2CH2CHCOOH
• 3-Ornithine (5C)
  CH2CH2CH2CHCOOH
• 4-Citrulline (5C1)
  CH2CH2CH2CHCOOH

I NH2
I NH2
I NH2
NH I
NH C- NH2
Guanido group
I NH2
I NH2
I NH2
NH I
O C- NH2
Ureido group
Ornithine and citrulline are not found in
proteins but are formed in urea cycle
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Aromatic a.as

• Phenylalanine CH2CHCOOH
• Tyrosine Parahydroxy phenylalanine
• 
  CH2CHCOOH

I NH2
I NH2
OH
13
Heterocyclic a.as

• Tryptophan CH2CHCOOH
• Histidine CH2CHCOOH
• Proline (imino acid)

I NH2
N
H
I NH2
NH
N
COOH
N
H
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PROTEIN DIGESTION
15

• .

Proteins
Parietal cells
Gastric HCL
1st
Denatured proteins
Chief cells
HOW?
Endopeptidase with broad Specificity (cleaves
the internal peptide bonds in which the
carboxylic group is of aromatic a.as or leucine)
Pepsinogen
Pepsin
Inactive zymogen
Active enzyme
then
Rennin in infants
Ca
Milk caseinogen
Soluble casein
Ca paracaseinate (milk clot)
Denaturation
Importance?
Large peptides some free a.as
Further digested by pepsin and other proteases
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• Large peptides some free a.as

Pancreatic endopeptidases including
Intestinal cells
Trypsinogen Trypsin
(inactive)
(active) Chymotrypsinogen
Chymotrypsin (inactive)
(active) Proelastase
Elastase (inactive)
(active) Collagenase
Cleaves peptide bonds in which the COOH is of
Basic a.as (arginine and lysine) Cleaves peptide
bonds in which the COOH is of Aromatic a.as or
leucine. Cleaves peptide bonds in which the
COOH is of small non polar a.as (a.as with
small uncharged R as glycine, alanine
serine) Catalyzes the hydrolysis of collagen
enteropeptidase
Smaller peptides free a.as
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Exopeptidases

• Smaller peptides free a.as

Pancreatic carboxypeptidases including
Cleaves the aromatic a.as from the C- terminal
end of the peptide Cleaves the basic a.as from
the C- terminal end of the peptide Cleaves
one a.a from the N- terminal end of the
peptide Cleaves a.as from the dipeptides or
tripeptides
Procarboxypeptidase A Carboxy
peptidase A (inactive)
(active) Procarboxypeptidase B
Carboxy peptidase B (inactive)
(active)
Trypsin
Intestinal proteases
Aminopeptidases Dipeptidases and tripeptidases
a.as
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AMINO ACIDS ABSORPTION
19
In infants, Ig A in the clostrum of milk is
absorbed without digestion by pinocytosis
giving
immunity to the babies
Intestinal cell
Intestinal lumen
Blood
IgA IgA
IgA IgA
gA
gA
gA
20

• Aa.s resulting from protein digestion
• are absorbed from the small intestine by
• passive transport mechanism (For D-aas).
• Active transport mechanism (For L-aas and
  dipeptides)
• ? Carrier protein transport system
• ( sodium amino acid carrier system ).
• ? Glutathione transport system
• (?-glutamyl cycle)

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Carrier protein transport system ( sodium
amino acid carrier system )
Intestinal lumen
K
Na
a.a.
a.a.
Na
ADPPi
Cell membrane
Na/K ATPase
Carrier
ATP
Intestinal cell
a.a.
Na
K
Cytoplasm
Portal blood
a.a.
22

• This system transport the a.a. against its conc.
  gradiant using energy derived from Na/K pump.
• Here a.as are absorbed by specific carrier
  protein in the cell membrane of the small
  intestinal cells.This carrier protein has one
  site for the a.a. and another site for the Na.
  It transports them from the intestinal lumen
  across the cell membrane to the cytoplasm. Then
  the a.a. passes to the blood down its conc.
  Gradient, while the Na is pumped out from the
  cell to the intestinal lumen by Na/K pump
  utilizing ATP as a source of energy derived from
  Na/K pump.

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Glutathione transport system
(?-glutamyl cycle)
R
a.a.
NH2CHCOOH
?-glutamyl transpeptidase(transferase) (GGT)
Cell membrane
CO-NH-CH-COOH
CH2
R
?-glutamyl- cysteinylglycine
cysteinylglycine
CH2
CH-NH2
ADPPi
dipeptidase
glutathione synthetase
COOH
?-glutamyl a.a.
ATP
glycine
?-glutamyl- cysteine
?-Glutamyl cyclotransferase
a.a.
ADPPi
?-Glutamylcysteine synthetase
cysteine
5-oxoproline
ATP
H2C CH2
Glutamic acid
Oxoprolinase
H2O
CH-COOH
OC
N
ATP
ADPPi
I H
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• ?This transport system is for
• the transport of a.a.s from the
  extracellular space to the cytoplasm in the
  intestine,kidney, brain liver(bile ductule
  cells)
• 
  So it is
  not important only for the uptake of a.as from
  the intestinal lumen (a.a. absorption) .
• 
  
• ? 3 ATP molecules are utilized for the transfer
  of one a.a.
• ?Clinical notes
• The blood conc. of GGT enzyme is increased
  in cholestasis chronic alcholism ( so used as a
  liver function test).
• Oxoprolinuria Inherited deficiency of
  glutathione synthetase enzyme , leading to
  increase levels of 5 oxoproline in blood urine
  acidosis neurological damage

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Quiz

• The basis of allergic reactions to
  food

26

• Fate of absorbed a.as

Enter in the formation of a.a. pool
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AMINO ACID POOL
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? Definition It include the free a.as
distributed throughout the body - The a.a.
pool contains 100 gm a.as.50 of these a.as are
in the form of glutamate glutamine
(Why?) - In contrast to the amount of
protein in the body (about 12 Kg in 70 Kg man),
the a.a. pool is small (only 100
gm) ?Sources fate of the a.a. pool
Non essential a.a.s synthesized in the body
Diatery proteins
Tissue proteins
a.a.s
Amino acid pool
Catabolism(Deamination)
Anabolism
Synthesis of
Fate of deamination products
Proteins
Other nitrogenous compounds
a- keto acid
Ammonia
?Aminosugars ?Nitrogenous bases of
phospholipids ?Purines pyrimidines ?Neurotransmi
tters ?Niacin ?Creatine ?Heme
?Tissue proteins ?Plasma proteins ?Enzymes ?Some
hormones ?Milk
Krebs cycle
Ketone bodies
Synthetic pathway
Catabolic pathway
glucose
CO2H2O ENERGY
Excreted in urine
Non essential a.a.synthesis
glutamine
Urea
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AMINO ACID CATABOLISM
30

• Catabolism of the a.a.s occurs by
• Deamination (removal of the amino group)
• either by
• General methods of deamination
• ?Oxidative deamination
• ?Transamination
• ?Transdeamination
• Or
• Specific methods of deamination
• For certain a.a.s

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Oxidative deamination

• ? Definition It is the
• oxidation (removal of hydrogen)
• and deamination (removal of the amino group
  which is liberated as free ammonia)
• giving
• a- ketoacid and ammonia (reversible
  reactions)
• R C COOH

H
NH3
l NH2
l l O
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? Site In most tissues , mostly in the liver
and kidney

• ? Enzymes involved
• 1- L-glutamate dehydrogenase enzyme
  Present in the cytosol
  mitochondria of most tissues
• HOOCCH2CH2CHCOOH ?
  ? HOOCCH2CH2C COOH

NAD(P)
NAD(P)HH
H2O
NH3
l l O
L-GLUTAMATE DEHYDROGENASE ENZYME
l NH2
L-glutamic acid
a- ketoglutaric acid
Coenzyme is NAD or NADP
33

• Regulation The direction of the reaction depends
  on
• 1- Availability of the substrates
• --Relative conc. Of (a-ketoglutarate NH3)
  and (glutamate).
• --Ratio of NADP NADPHH
• 2- Allosteric regulation
• --Activators ADP or GDP.
• -- Inhibitors ATP ,GTP NADH

34
QUIZ

• ---After a protein meal , in which direction the
  reaction proceeds? Why?
• ---After a CHO meal ,
• in which direction the
• reaction proceeds?
• Why?

35

• 2- D- L- a.a. oxidases
• Present only in the liver and kidney in
  minimal amounts
• They are of low activity in the mammalian
  tissue
• N.B. a.a.s L-a.as mammalian
  proteins are formed of only L-a.as
• D-a.as are found
  in plants and the cell wall of
  
  
  microorganisms but not used in the synthesis of
  mammalian proteins
• L-a.a. oxidase It deaminates most of the
  naturally occuring a.a.s.
• R CH COOH
  R C COOH

NH3
H2O
L-a.a. oxidase
l NH2
l l O
FMN
FMNH2
L- amino acid
a- keto acid
O2
H2O2
H2O2
Catalase
2 H2O O2
36

• D-a.a. oxidase It deaminates D-a.as present in
  diet
• giving a-
  keto acids
• that either transaminated to the
  coressponding L-a.as
• or converted to
  glucose or F.as
• or catabolized to CO2
  H2O energy
• R CH COOH
  R C COOH

H2O
NH3
D-a.a. oxidase
l NH2
l l O
FAD
FADH2
D- amino acid
a- keto acid
O2
H2O2
L- amino acid
Transaminase
H2O2
Catalase
a- keto acid
2 H2O O2
Glucose f.as CO2H2O
R CH COOH
l NH2
L- amino acid
37
?Importance of oxidative deamination

• L-glutamate dehydrogenase enzyme is the only a.a.
  that undergoes oxidative deamination in the
  mammalian tissue.
• Oxidative deamination by L- glutamate
  dehydrogenase is an essential component of
  transdeamination.
• So it is important in deamination of
  most a.as.

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Transamination

• ?Definition It is the transfer of amino group
• from one a- a.a. to a- keto acid
  
• to form a new a- a.a a new a-
  keto acid (reversible
  reaction)
• ? Enzymes involved Transaminases or
  aminotransferases
  Coenzyme PLP (Pyridoxal phosphate)

R C H COOH l NH2
R C COOH l l O
?
?
Transaminase PLP
?
?
R C H COOH l NH2
R C COOH l l O
39

• ?Site In the cytosol or both the cytosol the
  mitochondria of most cells especially the liver
• ? All a.as except threonine,lysine,prolinehydroxy
  proline may undergo transamination

40
? a-ketoglutarate glutamate are often involved
in transamination reactions
a- a.a.
a- keto glutarate
?
?
Glutamate transaminase
?
?
a- keto acid
Glutamic acid
41

• ? Transaminases of clinical importance are
• Alanine transaminase(ALT)
• Glutamate pyruvate transaminase(GPT)
• Glutamic acid Pyruvic acid
  Alanine a- keto glutarate
• Aspartate transaminase(AST)
• Glutamate oxaloacetate transaminase(GOT)
• Glutamic acid Oxaloacetic acid
  Aspartic acid a- keto glutarate

ALT
PLP
CH3 C COOH l l O
CH3 C H COOH l NH2
ALT
PLP
HOOCCH2CHCOOH l NH2

HOOCCH2CCOOH l l O

42

• ?Value of transamination
• Function
• 1- Degradation of a.as to form a-
  keto acids.
• 2- Synthesis of non essential
  a.as from CHO.
• Diagnostic value
• Transaminases are normally intracellular
  enzymes. They are elevated in the
  blood when damage to the cells producing
  these enzymes occurs.
• Increase level of both ALT AST
  indicates possible damage to
  the liver cells.
• Increase level of AST alone suggest
  damage to heart muscle ,skeletal
  muscle or kidney.

43
Transdeamination

• ?Definition
• It is the combination of transamination
  oxidative deamination.
• It includes the transamination of most a.as with
  a keto glutarate to form glutamate then the
  glutamate is oxidatively deaminated reforming a
  keto glutarate and giving ammonia. This provides
  a pathway by which the amino group of most a.as
  is released in the form of ammonia.

?
a- keto glutarate
?
a- a.a.
?
?
NH3
?
NAD(P)HH
Glutamate transaminase
L-GLUTAMATE DEHYDROGENASE ENZYME
NAD(P)
?
H2O
?
a- keto acid
Glutamic acid
?
?
44
On biochemical basis explain

• Removal of ammonia from a.as can not be
  explained alone by transamination nor by
  oxidative deamination alone
• It can not be explained by transamination alone
  as no free ammonia is liberated nor by oxidative
  deamination alone as oxid. Deamination works
  efficiently only on glutamic acid as L- glutamate
  dehydrogenase is of high activity in the
  mammalian tissue,while the L- amino acid oxidase
  which works on most a.as is of low activity.
• The formation of NH3 from a a.as occurs mainly
  via the a amino nitrogen of glutamate
• As this occurs by transdeamination, and L-
  glutamate is the only a.a that undergoes
  oxidative deamination at an appreciable rate in
  the mammalian tissue.

45

• Another importance of transdeamination is we can
  form a a.a. from ammonia

?
a- keto glutarate
?
a- a.a.
?
?
NH3
?
NAD(P)HH
Glutamate transaminase
L-GLUTAMATE DEHYDROGENASE ENZYME
NAD(P)
?
H2O
?
a- keto acid
Glutamic acid
?
?
46
Specific methods of deamination

• 1- L- glutamate dehydrogenase
• Said before

47

• 2- Glycine oxidase
• as the mechanism of action of D- amino acid
  oxidase
• CH2 COOH
  CH COOH

H2O
NH3
Glycine oxidase
l l O
l NH2
FAD
FADH2
Glycine
Glyoxylic acid
O2
H2O2
H2O2
Catalase
2 H2O O2
48

• 3- Glycine cleavage system
• FH4
  CH2-FH4 NH3 CO2

NAD
NADHH
CH2 COOH NH2
Tetrahydrofolic acid
Methylene tetrahydrofolic acid
Glycine
49

• 4- Histidase ( Non oxidative deamination)

CH2 CH COOH
NH2
Histidase
CH CH COOH
N
NH
NH3
N
NH
Urocanic acid
Histidine
50

• 5-Dehydratases (Non oxidative deamination)
• For hydroxy containing a.as (serine
  threonine)

CH2CHCOOH l l OH NH2
CH2CCOOH l NH2
CH3CCOOH ll NH
Serine dehydratase
PLP
H2O
Serine
aiminopropionic acid
aaminoacrylic acid
H2O
Serine dehydratase
NH3
CH3CCOOH ll O
Pyruvic acid
CH3CHCHCOOH l l
OH NH2
CH3CH2CCOOH ll
O
Threonine dehydratase
PLP
NH3
aketobutyric acid
Threonine
51

• 6-Desulfhydrases
• The a.a. cysteine is deaminated by cysteine
  desulfhydrase

CH2CHCOOH l l SH NH2
CH2CCOOH l NH2
CH3CCOOH ll NH
Cysteine desulfhydrase
PLP
H2S
Cysteine
aaminoacrylic acid
aiminopropionic acid\
H2O
Cysteine desulfhydrase
NH3
CH3CCOOH ll O
Pyruvic acid
52

• 7-Hydrolytic deaminases Deamination by H2O
• Glutaminase asparginase which catalyze the
  hydrolytic deamination of glutamine
  aspargine respectively.

NH3
H2O
HOOCCH2CH2CHCOOH
l NH2
OCCH2CH2CHCOOH l
l NH2 NH2
Glutaminase (in kidney, intestine)
Glutamic acid
Glutamine
53

• 8-Reductive deaminases
• By the action of intestinal bacteria
  on the a.as
  (putrefaction) with the production of the
  corresponding organic
  acids.

NH3
2H
R C H2 COOH
R C H COOH l NH2
Corresponding organic acid
a- a.a.
54

• Deamination products

a- keto acid
NH3
55
AMMONIA METABOLISM
56

• Blood level Normally lt 0.1 mg / dl
• Urine level 0.7 gm / day
• Sources fate

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Amino acids
Various nitrogenous compounds as
Purines pyrimidines (amino groups attached to
the rings)
Some neurotransmitters as
Deamination --Oxidative deamination --Transdeami
nation --Specific deamination Methods ( MAIN
SOURCE)
Urea secreted into the intestine
Monoamines --serotonin epinephrine,norepinephri
ne, dopamine their metabolites metanephrine,
normetanephrine 3 methoxy tyramine
Histamine
Catabolism
Histaminase
Intestinal bacterial urease
Monoamine oxidase (MAO)
NH3
Synthetic pathway
Catabolic pathway

Liver (MAIN FATE)
Transdeamination
Non essential a.a.synthesis
glutamine
Urea
Excreted in urine
Urine
58

• ?Urea formation is the main pathway by which the
  body gets rid of NH3
• ?Glutamine formation
• --By the glutamine synthetase enzyme which is a
  mitochondrial enzyme
• Glutamic acid ammonia
  glutamine
• -- Glutamine is produced in many extra renal
  tissues esp. important
• In the muscle
• In the liverThe formation of glutamine can
  be considered as a mechanism for scavenging NH3
  that has not been incorporated into urea.
• In the brain It removes the toxic effect of
  NH3 in the brain .Then the glutamine goes via the
  blood to the kidneys where it become hydrolyzed
  by glutaminase into glutamic acid and NH3 which
  is excreted in urine,(This accounts for 60 of
  the NH3 excreted in urine)
• Glutamine
  Glutamic acid

glutamine synthetase
glutaminase
H2O
NH3
59

• ?NH3 produced from a.a. deamination in the kidney
  is directly
• excreted in urine ( This acconts for 40of NH3
  excreted in urine)
• N.B. NH3 produced from a.a deamination in the
  kidney esp.
• glutamine regulates acid base balance preserve
  cations

60
UREA CYCLE
61
CO2
NH3
Steps
Cytoplasm
Mitochondria
NH3
CO2

2ATP
Carbamoyl-phosphate synthetase 1
2 ADP1 Pi
O O ll
ll H2N C O P OH
l OH
Carbamoyl-phosphate
CH2CH2CH2CHCOOH l
l NH NH2
l OCNH2
Pi
Ornithine transcarbamoylase
CH2CH2CH2CHCOOH l
l NH2 NH2
Citrulline
Ornithine
HOOCCH2CHCOOH l NH2

Citrulline
Ornithine
O ll
H2NCNH2
Aspartic acid
Urea
Arginino-succinate synthetase
Arginase
ATP
AMPPPi
CH2CH2CH2CHCOOH l
l NH
NH2 l NH2CNH
H2O
H2O

CH2CH2CH2CHCOOH
l l
NH NH2
l
NHCNH
l
HOOCCH2CHCOOH
Arginino succinate
Arginine
Arginino succinase
HOOCCH ll HCCOOH
Fumarate
62

• Carbamoyl- phosphate synthetase I is different
  from carbamoyl phosphate synthetase II

CPS II CPS I
Cytoplasm Mitochondria Site
Pyrimidine synthesis Urea synthesis Pathway
Nil N-acetyl glutamate ve effector
CTP Nil Inhibitor
Glutamine Ammonia Source of N
63
O ll
H2NCNH2

• Sources of the atoms of urea
• 1- C O from CO2
• 2- 1st N atom from NH3
• 3- 2nd N atom from aspartate

Glutamate is usually the immediate
precursor of both NH3
aspartate
Glutamate NH3
Aspartate
H2O
Oxaloacetate
NAD
a- ketoglutarate
Oxidative deamination
NADHH
Transamination
a- ketoglutarate
64

• Overall reaction
• NH3 CO2 Aspartate Urea
  fumarate
• There in no net gain or loss of ornithine,
  citrulline , argininosuccinate or arginine.
• Ornithine is regenerated with each turn of the
  urea cycle. The release of the high energy
  phosphate of carbamoyl phosphate as inorganic
  phosphate drives the reaction in the forward
  direction.

65
Fate of fumarate its link to TCA cycle
CO2
NH3
Oxaloacetate
Carbamoyl-phosphate
Malate
NADHH
NAD
3ATP
Fumarate
Ornithine
TCA CYCLE
Citrulline
Citrulline
Ornithine
Aspartate
Urea
Arginine
Arginino succinate
Fumarate
Transamination
Cytoplasmic fumarase
H2O
or
Malate dehydrogenase
Fasting state
Malate
Oxaloacetate
PEP
Glucose
Malate shuttle
NAD
NADHH
Fed state
NADP
Malic enzyme
NADPHH
Oxaloacetate
3ATP
ATP citrate lyase
Pyruvic acid
Pyruvic acid
Citrate
Citrate
Acetyl COA
f.a. synthesis
66

• Bioenergetics
• Urea cycle consumes four "high-energy" phosphate
  bonds (3 ATP hydrolyzed to 2 ADP and one AMP).
• 1 ATP ADP Pi
• 1 ATP ADP Pi
• 1 ATP AMP Pi Pi
• ?However. One NADHH molecule is produced by
  oxidative deamination of glutamate to NH3 and
  a-ketoglutarate. Glutamate provides the NH3 used
  in the initial synthesis of carbamoyl phosphate.
• ? Also fumarate in the cycle may be converted to
  malate in the cytosol . Malate then oxidized to
  oxaloacetate gives 1 NADHH equivalent to 3 ATP
  obtained from 3ADP,
• So the net energy expenditure is only one high
  energy phosphate .
• The two NADHH produced can provide energy for
  the formation of 5 ATP, a net production of one
  high energy phosphate bond for the urea cycle.
  However, if gluconeogenesis is underway in the
  cytosol, the latter reducing equivalent is used
  to drive the reversal of the glyceraldehyde 3-p
  dehydrogenase step instead of generating ATP. So
  the net energy expenditure is only one high
  energy phosphate .

Adenosine
P P P
67
Regulation

• Carbamoyl phosphate synthetase 1 is the key it
  has an absolute requirement for
• N-Acetylglutamic acid which act as an allosteric
  activa tor.
• The synthesis of N- Acetylglutamate from glutamic
  acid acetyl CO A is by
• high protein diet a.as especially arginine ?
  ?rate of urea cycle
• .

GLUTAMATE
Acetyl CO A
Acetate
a.as esp arginine
Synthetase
N- ACETYL GLUTAMATE
CO A
ALLOSTERICALLY carbamoyl phosphate synthetase
1
68
CREATINECREATININE
69

• Structure

Creatine (methyl guanido acetic acid)
CH2 COOH l
CH3 N
l HN C NH2

Creatinine CH2 CO
l CH3 N l
HN C NH
Phosphocreatine (Creatine phosphate)
CH2 COOH l
CH3 N
O l ll
HN C NH P OH
l
OH
70
Synthesis of creatine creatine phosphate
their degradation into creatinine
Creatine is formed from 3 a.as ( glycine,
arginine methionine )
Glycine transamidinase

• Arginine

Glycine
Ornithine
Guanidoacetic acid


(kidney, pancreas)
CH2 COOH NH2

CH2CH2CH2CHCOOH l
l NH
NH2 l NH CNH2
CH2 COOH
l
NH l
HN C NH2
CH2CH2CH2CHCOOH l
l NH2
NH2
S- adenosyl methionine
Methyl transferase (liver)
S- adenosyl homocysteine
CH2 COOH
l CH3
N l HN
C NH2
Creatine phosphokinase
Creatine (methyl guanido acetic acid)

Creatine phosphate
(liver, brain, muscle)
CH2 COOH l
CH3 N O
l ll HN C NH P OH
l
OH
Non enzymatic (spontaneous)
Rapid Pi
Slow H2O
CH2 C O l
CH3 N l
HN C N H
Creatinine
Excreted in urine
71

• Importance of creatine
• ?Creatine is converted to creatine phosphate
  using ATP as a phosphate donor. Creatine
  phosphate acts as a store of high energy
  phosphate ,so it is used as a source of energy
  in time of need (as it can give the phosphate
  group to ADP to form ATP).
• ?Creatine is present mainly in the muscle (98) ,
  but also in the brain blood . Both the muscle
  the brain contain large amounts of creatine
  phosphate.
• ?During muscular relaxation,creatine becomes
  converted to creatine phosphate to be used as a
  source of energy during muscular contraction.
• (During ms contraction, ATP is mostly derived
  from glycolysis for which ms glycogen acts as a
  substrate,However before ATP from glycolysis
  become available, phosphocreatine acts as a
  temporary source of ATP.So it is especially
  important during the early stages of ms
  exercise(1st few minutes),where the largest
  quantities of creatine P are found ).
• Level of creatine in the blood urine
• ?Plasma creatine level 0.4 mg
• Whole blood creatine level 2 - 4 mg
• ? Normally little creatine is excreted in urine (
  normally lt 100 mg /day ) .This is more in females
  children due to low androgens which increase
  the uptake of creatine by ms.

72

• ?Def. It is the presence of large
  amounts of creatine in urine
• (Normal level of creatine
  excreted in urine is lt 100 mg / day.)
  
• ?Causes
• 1- Physiological creatinuria
• in children due to -
  ?androgens ? ? uptake of creatine by the muscle
• 
  - small mass of the ms
• in female due to - ? free
  androgen especially during pregnancy.
• -
  after labour due to involution of the uterus.
• 2- Pathological creatinuria
• in males with ? androgens(
  hypogonadism )
• in muscle atrophy as in
  myopathies.
• ? protein catabolism in
  --hormonal disturbances as D.M. ,
• hyperthyrodism and cushing
  syndrome,--also in fever and starvation
  

Creatinuria
73

• Creatinine levels in serum urine
• ?Normal serum creatinine level 0.5 - 1.5 mg.
• Causes 1- Impairment of kidney function about
  50 of kidney function must be
  
• lost before serum creatinine
  is ?.
• 2- ? ms mass as acromegally
  gigantism.

? serum creatinine level
? serum creatinine level
74

• ? Normal urine creatinine level 1 - 1.5 mg .
• It is more in male due to greater ms mass ----
  male 1.5 g / day.
• 
  ---- female 1 gm / day.
• There is a steady production of constant amount
  of creatinine that is proportional to the total
  amount of phosphocreatine creatine in the body
  , which is in turn proportional to the ms mass
  of the individual .
• So, the serum urine creatinine is
  constant for an individual approximately
  proportional to the ms mass.
• Creatinine excretion rate (creatinine
  coefficient)
• It is the amount of creatinine measured in
  mg / kg body weight / day.
• It is said to be remarkably constant (21 in
  male 16 in female).(Why?)
• So, creatinine excretion rate (creatinine
  coeffecient) can be used to check the
• accuracy of 24 hr collection of urine.
  If found lower than expected, it
• indicates that part of the urine was
  discarded .

75

• Creatinine clearance
• The volume of serum or plasma that would be
  cleared of creatinine by one minute's excretion
  of urine. value that reflects the body's ability
  to excrete creatinine it is used to diagnose and
  monitor renal function.
• Urinary creatinine is 100 endogenous in
  origin i.e. not affected by diet being
  synthesized in the body no diurnal variation
  .So it can be used for calculation of urinary
  output of substance amount of creatinine in
  urine.
• Also creatinine is chiefly filtered by the
  kidney, though a small amount is actively
  secreted. There is little-to-no tubular
  reabsorption of creatinine. If the filtering of
  the kidney is deficient, blood levels rise. As a
  result, creatinine levels in blood and urine may
  be used to calculate the creatinine clearance
  (CrCl), which reflects the glomerular filtration
  rate (GFR). The GFR is clinically important
  because it is a measurement of renal function.
  However, in cases of severe renal dysfunction,
  the creatinine clearance rate will be
  "overestimated" because the active secretion of
  creatinine will account for a larger fraction of
  the total creatinine cleared. Ketoacids,
  cimetidine and trimethoprim reduce creatinine
  tubular secretion and therefore increase the
  accuracy of the GFR estimate, particularly in
  severe renal dysfunction. (In the absence of
  secretion, creatinine behaves like inulin.)
• Creatinine clearance (CCr) can be calculated if
  values for creatinine's urine concentration
  (UCr), urine flow rate (V), and creatinine's
  plasma concentration (PCr) are known. Since the
  product of urine concentration and urine flow
  rate yields creatine's excretion rate, creatinine
  clearance is also said to be its excretion rate
  (UCrV) divided by its plasma concentration. This
  is commonly represented mathematically as
• Creatinine clearance(ml/min)

Urinary creatinine conc.X urine volume in
ml Plasma creatinine conc X time of
collection/min
76
NON PROTEIN NITROGENOUS COMPOUNDS
77

• NPN Compounds
• Def Are nitrogenous compounds not precipitated
  by protein ppting reagents.
• Include
• a.as NH3
• Urea Uric
  acid
• Creatine Creatinine

78
Urea Ammonia a.as
O ?? H2N C- NH2 NH3 R-CH CooH ? NH2 Structure
Co2. NH3. Aspartic acid Through the Urea cycle in liver. a.a. deamination (main source) other nitrogonous compounds as - purines pyrimidmes. - amines. -urea splitted in the intestine. Proteins in diet. Tissue protein catabolism. Synthesis of non essential a.as. (sources of a.a.pool) Source
Excretion in urine part Pass to intestine. Synthesis of a.as. Urea formation. Glutamine formation. Excretion in urine. Synthesis of Proteins as tissue Pr. Plasma Pr. Enzymes. Some hrs. Milk. Other nitrogenous comp as creatine,heme, neurotransmitters, purines pyrimidinet catabolism (deamination) giving - NH3 ? Fate said with NH3. - a -Keto-acids? glucose. ? ketone bodies. ? Co2 H2o energy. Fate
20-40 mg/dl lt 0.1 mg/dl. 4-6 mg/dl Blood level
20-40 gm/day. 0.7 gm/day 0.7 g/day Urine level
Bl. Urea is a krdnay function test ? in renal failure. Ammonia intoxication Amino acid uria Clinical implication
79
Uric acid Creatinine Creatine
Structure
Catabolism of purines (liver). -Creatine creatine phosphate. ( in ms, brain and other tissues.) - Arginine glycine methionine. ( in kidneys and liver.) Source
Excretion in urine. Excretion in urine. Uptake by ms and other tissues. Fate
2-7 mg/dl 0.5-1.5 mg 0.4 mg plasma 2-4mg blood Bl. Level
500 mg/day 1-1.5 gm/day lt 100 mg/day Urine level
Hyperuricemia (gout) Hypouricemia. ? serum creatinine - Renal failure. - ?ms mass as acromegally gigantism. ? serum creatinine in ? ms mass as ms dystrophy deblitation Creatinine coefficient for accuracy of collecting 24 hrs volume of urine. Creatinine clearance as measure of GFR. Creatinuria Clinical implications
CH2 C O l
CH3 N l
HN C N H
CH2 COOH l
CH3 N
l HN C
NH2
80

• Heterocyclic a.as
• 1- Tryptophan
• Synthesis essential a.a.
• Catabolism Both glucogenic and ketogenic.
• Tryptophan
• Tryptophan pyrrolase (oxygenase)
• N- formyl kynurenine
• Acetoacetic acid
• -keto adipic acida
• a-aminomuconate
• a-aminomuconate semialdehyde
• Quinolinic acid
• NADNADP
• Nicotinic acid
• 2-Acrolyl 3- amino fumarate
• 3-Hydroxyanthranilic acid
• Hydroxykynurenine
• Kynurenine