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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