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7/26/2019 Week 10 Lecture 1 Nitrogen Metabolism
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NITROGEN METABOLISM
Prof. Dr. Nazamid Saari
Department of Food ScienceUniversiti Putra Malaysia
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Learning outcomes
Recognize how amino acids/proteins are
turned into metabolic energy and the
chemical processes involved
Predict the energy content and value of the
chemical compounds
Identify its roles to human/animal as well as in
food production
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Nitrogen balance and amino acid
metabolism
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Nitrogen excretion
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INTRODUCTION
The biosynthesis of proteins requires acontinuous source of amino acids.
Amino acids are generated by the digestion of
proteins in the intestine or by the degradationof proteins within the cells.
Cellular proteins are constantly being
degraded and resynthesized. The short lived proteins usually play important
metabolic roles.
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Metabolic relationships of amino
acids
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Cont.
The pools (=amino acid available for metabolicprocess) of free amino acid in animals arederived from a combination of dietary sourcesand de novo synthesis.
Amino acids are important precursors of avariety of biological molecules as well as
providing the building blocks for polypeptideand protein synthesis. In addition, amino acidcarbons can be oxidized for energy productionafter removal of their amino group.
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Digestion of Dietary Proteins
Protein digestion begins in the stomach. The primary enzyme involved in proteolytic digestion is
pepsin which catalyzes the nonspecific hydrolysis of peptidebonds at an optimal pH of 2.
In the lumen of the small intestine, the pancreas secreteszymogens of trypsin, chymotrypsin, elastase ect
This battery of proteolytic enzymes breaks the proteinsdown into free amino acids as well as dipeptides andtripeptides.
The free amino acids as well as the di- and tri-peptides areabsorbed by the intestinal mucosa cells which subsequentlyare released into the blood stream where they are absorbedby other tissues.
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Protein digestion
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Turnover of Cellular Proteins
Cellular proteins are continually beingsynthesized and degraded cell.
Functional proteins are distinguished from oldproteins and are marked for degradation by theattachment ofan Ubiquitin tag.
Ubiquitin is a small protein found in alleukaryotic cells. Ubiquitin is attached to theterminal -amino group of lysine residues
marking these proteins for degradation. Three enzymes are involved in the tagging of a
protein. E1, The ubiquitin activating enzyme E2,ubiquitin conjugating enzyme E3,ubiquitin
protein ligase.
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Cont.
Once a protein is marked for degradation,proteasome executes the proteolysis using ATPto hydrolyze the peptide bonds of proteins.
The proteasome has a sedimentation coefficientof 26S and is composed of 2 subunits, a 20Sproteasome which contains all of the catalyticmachinery to digest proteins and a 19S regulatorysubunit.
The substrate proteins are degraded in aprocessive manner until the entire protein hasbeen reduced to peptides of 7 to 9 residues.
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Ubiquitin Related ProteinDegradation
Ubiquitin is a smallprotein(8.5 kD = 76amino acids)
Highly conserved amongall Eukaryotes.
When covalentlyattached to a protein,
ubiquitin marks thatprotein for destruction
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Tagging of Proteins
The carboxyl-terminal glycine of ubiquitincovalently attaches to -amino group oflysine residues on target protein
Requires ATP hydrolysis Three enzymes involved: 1) E1, ubiqutiin
activating protein, 2) E2, Ubiquitinconjugating enzyme, 3) E3, ubiquitin-protein ligase.
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Protein Ubiquitination
Multiple Ubiquitins can be polymerized to each other.
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What determines whether a proteinwill become ubiquinated?
E3 enzyme are readers of N-terminal amino acid residues
N-terminal amino acidsdetermine stability of protein
Also proteins rich in proline,glutamic acid, serine andthreonine (PEST sequences)
often have short lives. Other specific sequences (e.g.
cyclin destruction box) targetproteins for ubiquitination
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Ubiquitinated Proteins are Degradedby the 26S Proteosome
The 26S proteosome isa large proteasecomplex thatspecifically degrades
ubiquinated proteins 2 major components
20S proteosome core,19S cap.
Proteolysis occurs in20S domain
Ubiquitin recognitionoccurs at 19S domain
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26S Proteosome
ATP dependentprocess.
Protein is unfoldedas it enters 20Sdomain.
Ubiquitin notdegraded, butreleased andrecycled.
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Cont.
The peptide products are further degraded by
cellular proteases to yield the individual
amino acids.
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What is the fate of these amino acids?
The amino acids that are produced are eitherutilized for the biosynthesis of newer proteins ordegraded.
In mammals, amino acids are degraded in theliver by deamination of amino acids to form -ketoacids.
The - ketoacids are metabolized and the
remaining carbon skeletons enters themetabolic mainstream as precursors forgluconeogenesis or as citric acid cycleintermediates.
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LIVER
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PYRIDOXAL PHOSPHATE
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Three stages of amino acid catabolism
1. Deamination (removal of the -amino
group and transport to the liver)
2. Urea synthesis (to excrete nitrogen; occurs
only in the liver)
3. Conversion of the carbon skeleton to one of
seven metabolic intermediates
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Deamination/Transamination By Transaminase/Amino
Transferase (common name)
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Cont..Transamination of Amino Acid
There are three main transaminases or Amino transferases,all requiring Pyridoxal-P derived from vitamin B6(pyridoxine) via phosphorylation as a cofactor:
Glutamate aminotransferase (third most active in liver):amino acid + 2-oxoglutarate/-ketoglutarate 2-oxoacid/-keto acid + glutamate
Alanine aminotransferase (second most active in liver): ala+ 2-oxoglutarate/-ketoglutarate pyruvate +
glutamate Aspartate aminotransferase (most active in liver): asp + 2-
oxoglutarate/-ketoglutarate oxalacetate +glutamate
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ContTransamination of Amino Acid
The various aminotransferases in the liver all
funnel excess N to glutamate and aspartate.
Glutamate can then be deaminated by
Glutamate dehydrogenase to give ammonia,
contributing up to 1/2 of the N in urea.
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Ammonia production
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Cont.
Most transaminases share a common
substrate and product (glutamate and
oxoglutarate) with glutamate dehydrogenase,
and this permits a combined nitrogenexcretion pathway for individual amino acids
that is commonly described as TRANS-
DEAMINATION. This process demonstrates thecentral roles of glutamate in the overall
control of nitrogen metabolism
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Amino group transport
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Urea Cycle
Every amino acid contains at least one aminogroup. Amino acid catabolism generatesammonia which is sensitive to brain tissue.
Therefore every amino acid degradation pathway
has a key step where the amino group isremoved.
Cells get rid of excess ammonia by the reductiveamination of ketoglutarate to form glutamate
by glutamate dehydrogenase and the conversionof glutamate into glutamine by glutaminesynthetase
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Cont.
-Ketoglutarate + NH4+ + NADH Glutamate + NAD+
Glutamate + NH4+ +ATP Glutamine + ADP + Pi
Glutamate is a neurotransmitter. Glutamate is also theprecursor for -aminobutyrate (GABA) which is another
important neurotransmitter. High concentrations of ammonia
deplete the concentration of glutamate which produces a
similar decrease of GABA which impairs brain function.
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UREA CYCLE
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Urea Cycle
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Cont
step1 ornithine transcarbamylase catalyzes
carbamoyl phosphate to transfer the
carbamoyl group to ornithine (non-standard
aa) to form citrulline (non-standard aa) takesplace in mitochondria; citrulline transported
out of mitochondria in exchange for ornithine
source of first N in urea
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Cont.
step 2 argininosuccinate synthetase
condenses citrulline with aspartate as source
of second N in urea to form arginosuccinate
requires hydrolysis of ATP to PPi and then to2Pi takes place in cytoplasm step 3 carbon
skeleton of aspartate removed as fumarate by
argininosuccinase arginine is producedtakes place in cytoplasm
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Cont.
step 4 urea is formed from arginine by
arginase and ornithine regenerated ornithine
is transported
Urea/TCA cycle coupling (Krebs
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Urea/TCA cycle coupling (Krebs
bicycle)
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cont
The urea cycle and the tricarboxylic acid cycle arecoupled together through fumarate andaspartate. Thus unless the fumarate releasedwhen arginosuccinate is cleaved can be cycled
through the TCA cycle to oxaloacetate, the ureacycle will be slowed or inhibited. Fumarate isthe precursor to oxaloacetate Oxaloacetatecan: be transaminated to aspartate and feed
back into urea cycle condense with AcCoA andfeed into citric acid cycle proceed intogluconeogenesis be converted to pyruvate
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Amino acid carbons
Glucogenic (Aspartic acid, glutamic acid, asparagine,glutamine, histidine, proline, arginine, glycine, alanine,serine, cysteine, methionine, valine) and Ketogenic(leucine and lysine) Amino Acids. Both Glucogenic and
Ketogenic (phenylalanine, tyrosine, tryptophan,isoleucine, and threonine)
The carbon skeletons of amino acids are metabolized,resulting in intermediates which are central to either
carbohydrate or lipid metabolism. Those which aremetabolized to yield potential substrates forgluconeogenesis are termed glycogenic, those whichyield acetate or acetoacetate are termed ketogenic.Some amino acids yield both kinds of intermediate.
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Amino acid carbon metabolism
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Cont.
This panel represents central carbon
metabolism and the points at which various
amino acid structures feed into it. Note that
some amino acids may feed differentmetabolic products into this scheme at two
different points if the carbon skeleton is
metabolized to produce two different kinds offragments (i.e. some amino acids can be both
glycogenic and ketogenic)
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Metabolic intermediates
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Summary
Synthesis of UREA requires energy input as
follow:
CO2+NH4+ + 3ATP + aspartate +2H2O ----
Urea + 2ADP +2Pi +AMP +PPi + Fumarate
Formation of one molecule of UREA requires theenergy from cleavage of 4 phosphoanhydride
bonds
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cont.
Step 1: 2 ATP---2ADP + Pi
Step 3: ATP---AMP + Ppi
Followed by
. Ppi + H2O ---2Pi