Non Protein AminoAcids.

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NON PROTEIN AMINOACIDS

• M.PRASAD
• MSC Medical BIO-CHEMISTRY,
• Ph.D. Research scholar

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Non protein amino acids

• These amino acids, although never found in
  proteins, perform several biological important
  function.
• These NPAAs and D-AA speculated to be related to
  auto immune disease and to aging

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• understand the role of NPAAs and D-AA s in
  auto immune disease and aging, the determination
  of these NPAAs and D-AA s is required.
• any nonprotein amino acid can be chemically
  incorporated into peptides, provided that
  appropriate methods are designed for protecting
  the functional group.

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• Nonprotein amino acids with no cytotoxicity have
  been known to be incorporated into proteins. For
  examples,
• tyrosine and tryptophan residues in some proteins
  have been substituted with m-fluorotyrosine and
  4-fluorotryptophan respectively, without any
  effects on the protein functions, and the 19F
  nuclei have been used as magnetic resonance

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• One NPA that has received some attention is
  canavanine, (L-2-amino-4-(guanidinooxy)butyric
  acid), the guanidinooxy structural analogue of
  arginine.
• These non protien amino acids are classified as
  alpha and non alpha amino acids
• Alpha amino acids
• a) ornithine
• b) citruline
• c) arginosuccinic acid
• d) thyroxine
• e) triodothyroxine
• f) S-Adenosylmethionine
• g) Homocysteine
• h) 3,4-Dihydroxy phenylalanine (
  DOPA)
• I ) creatinine
• j) ovathiol
• k) Azaserine

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• 2) NON ALPHA amino acids
• a) beta alanine
• b) beta aminoisobutyric acid
• c) gama aminobutyric acid(GABA)
• d) aminolevulinic acid (ALA)
• e) taurine

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Alpha - aminoacids

• 1) ornithine
• ornithine is precursors of polyamine
• Hydrolytic cleavage of the guanidino group of
  arginine, catalyzed by liver arginase, releases
  urea .
• The other product, ornithine, reenters liver
  mitochondria and participates in additional
  rounds of urea synthesis.
• Ornithine and lysine are potent inhibitors of
  arginase, and compete with arginine.
• Arginine also serves as the precursor of the
  potent muscle relaxant nitric oxide (NO) in a
  Ca2-dependent reaction catalyzed by NO synthase
• 2) Citrulline
• Citrulline is intermediates in the biosynthesis
  of urea

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• L-Ornithine transcarbamoylase catalyzes transfer
  of the carbamoyl group of carbamoyl phosphate to
  ornithine,forming citrulline orthophosphate
  While the reaction occurs in the mitochondrial
  matrix, both the formation of ornithine and the
  subsequent metabolism of citrulline take place in
  the cytosol.
• Entry of ornithine into mitochondria and exodus
  of citrulline from mitochondria therefore involve
  mitochondrial inner membrane transport systems

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3)Arginosuccinic acid

• Arginosuccinic acid is intermediates in the
  biosynthesis of urea
• Argininosuccinate synthetase links aspartate and
  citrulline via the amino group of aspartate and
  provides the second nitrogen of urea.
• The reaction requires ATP and involves
  intermediate formation of citrullyl-AMP.
  Subsequent displacement of AMP by aspartate then
  forms arginosuccinate.

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In addition to patients that lack detectable
argininosuccinate synthetase activity a 25-fold
elevated Km for citrulline has been reported. In
the resulting citrullinemia, plasma and
cerebrospinal fluid citrulline levels are
elevated, and 12 g of citrulline are excreted
daily.
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4) Thyrosine and triodothyroxine

• Tyrosine forms norepinephrine and epinephrine,
  and following iodination the thyroid hormones
  triiodothyronine and thyroxine.
• Use of measurement of blood thyroxine or
  thyroid-stimulating hormone (TSH) in the neonatal
  diagnosis of congenital hypothyroidism.
• The amino acid tyrosine is the starting point in
  the synthesis of the catecholamines and of the
  thyroid hormones tetraiodothyronine (thyroxine
  T4 ) and triiodothyronine (T3)

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• thioredoxin reductase, glutathione peroxidase,
  and the deiodinase that converts thyroxine to
  triiodothyronine.
• The clinical history, physical examination, and
  lab results were all consistent with primary
  hypothyroidism. Accordingly, the patient was
  started on a low dose of thyroxine (T4 ).
• It is important to begin therapy with a small
  dose of T4, as larger doses can precipitate
  serious cardiac events, due to the changes in
  metabolism caused by administration of the
  hormone.
• Thyroxine (T4), free 4.0 pmol/L (normal
  10.321.9 pmol/L)

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5)S-Adenosylmethinine homocysteine

• S-Adenosylmethionine, the principal source of
  methyl groups in metabolism, contributes its
  carbon skeleton to the biosynthesis of the
  polyamines spermine and spermidine.
• Homocystinuria
• Homocystinuria Cystathionine -synthase Lens
  dislocation,
• thrombotic vascular disease, mental retardation,
  osteoporosis AR  
• Homocystinuria
• 5,10-Methylenetetrahydrofolate reductase
• Mental retardation, gait and psychiatric
  abnormalities, recurrent strokes ,Mental
  retardation, hypotonia, seizures, megaloblastic
  anemia

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• Pathways, enzymes, and coenzymes involved in the
  homocystinurias. Methionine transfers a methyl
  group during its conversion to homocysteine.
• Defects in methyl transfer or in the subsequent
  metabolism of homocysteine by the pyridoxal
  phosphate (vitamin B6)-dependent cystathionine
  b-synthase increase plasma methionine levels.
• Homocysteine is transformed into methionine via
  remethylation. This occurs through methionine
  synthase, a reaction requiring methylcobalamin
  and folic acid.
• Deficiencies in these enzymes or lack of
  cofactors is associated with decreased or normal
  methionine levels. In an alternative pathway,
  homocysteine can be remethylated by
  betainehomocysteine methyl transferase

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• Life-threatening vascular complications
  (affecting coronary, renal, and cerebral
  arteries) can occur during the first decade of
  life and are the major cause of morbidity and
  mortality.
• Classic homocystinuria can be diagnosed with
  analysis of plasma amino acids, showing elevated
  methionine and presence of free homocystine.

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• Total plasma homocysteine is also extremely
  elevated (usually gt100 M). Treatment consists of
  a special diet restricted in protein and
  methionine and supplemented with cystine.
• In approximately half of patients, oral
  pyridoxine (25500 mg/d) produces a decrease in
  plasma methionine and homocystine concentration
  in body fluids.
• Folate and vitamin B12 deficiency should be
  prevented by adequate supplementation. Betaine is
  also effective in reducing homocystine levels.

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6) 3-4 Dihydrophenylalanine(DOPA)

• Neural cells convert tyrosine to epinephrine and
  norepinephrine. While dopa is also an
  intermediate in the formation of melanin,
  different enzymes hydroxylate tyrosine in
  melanocytes.
• Dopa decarboxylase, a pyridoxal
  phosphate-dependent enzyme, forms dopamine.
  Subsequent hydroxylation by dopamine -oxidase
  then forms norepinephrine.
• In the adrenal medulla, phenylethanolamine-N-methy
  ltransferase utilizes S-adenosylmethionine to
  methylate the primary amine of norepinephrine,
  forming epinephrine .
• Tyrosine is also a precursor of triiodothyronine
  and thyroxine.
• DOPA ... related to dopaminerelationship to
  Parkinson's Disease

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• Dopamine, Norepinephrine, and Epinephrine
• 1. SYNTHESIS OF THE CATECHOLAMINE
  NEUROTRANSMITTERS
• These three neurotransmitters are synthesized in
  a common pathway from the amino acid L-tyrosine.
• The first and rate-limiting step in the synthesis
  of these neurotransmitters from tyrosine is the
  hydroxylation of the tyrosine ring by tyrosine
  hydroxylase, a tetrahydrobiopterin(BH4)-requiring
  enzyme. The product formed is dihydroxyphenylalani
  ne or DOPA.
• The phenyl ring with two adjacent OH groups is a
  catechol, andhence dopamine, norepinephrine, and
  epinephrine are called catecholamines.

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• The second step in catecholamine synthesis is the
  decarboxylation of DOPA to form dopamine. This
  reaction, like many decarboxylation reactions of
  amino acids,equires pyridoxal phosphate.
• Dopaminergic neurons (neurons using dopamine as
  a neurotransmitter) stop the synthesis at this
  point, because these neurons do not synthesize
• the enzymes required for the subsequent steps.
  Neurons that secrete norepinephrine synthesize it
  from dopamine in a hydroxylation reaction
  catalyzed by dopamine -hydroxylase (DBH). This
  enzyme is present only within the storage
  vesicles of these cell
• Although the adrenal medulla is the major site of
  epinephrine synthesis, it is also synthesized in
  a few neurons that use epinephrine as a
  neurotransmitter.

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• 7)Creatinine
• Creatinine is formed in muscle from creatine
  phosphate by irreversible, nonenzymatic
  dehydration and loss of phosphate.
• Since the 24-h urinary excretion of creatinine
  is proportionate to muscle mass, it provides a
  measure of whether a complete 24-h urine specimen
  has been collected.
• Glycine, arginine, and methionine all participate
  in creatine biosynthesis. Synthesis of creatine
  is completed by methylation of guanidoacetate by
  S-adenosylmethionine.

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Normal values

• Creatinine 200 mol/L (4480 mol/L)
•   Female -- 4480 mol/L 0.50.9 ng/mL
• male -- 53106 mol/L 0.61.2 ng/mL

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8) Ovathiol-

• Sulfur containing amino acid found in fertilized
• Eggs, and acts as an antioxidant
• 9) Azaserine (antibiotic)
• Purine deficiency states, while rare in humans,
  generally reflect a deficiency of folic acid.
• Compounds that inhibit formation of
  tetrahydrofolates and therefore block purine
  synthesis have been used in cancer chemotherapy.
• Inhibitory compounds and the reactions they
  inhibit include azaserine, diazanorleucine,
  6-mercaptopurine , and mycophenolic acid .

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• II) Non--Amino Acids
• Non--amino acids present in tissues in a free
  form include -alanine, -aminoisobutyrate, and
  -aminobutyrate (GABA). -Alanine is also present
  in combined form in coenzyme A and in the
  -alanyl dipeptides carnosine, anserine and
  homocarnosine .
• 1) Beta-Alanine -Aminoisobutyrate
• Alanine and -aminoisobutyrate are formed during
  catabolism of the pyrimidines uracil and thymine,
  respectively . Traces of -alanine also result
  from the hydrolysis of -alanyl dipeptides by the
  enzyme carnosinase. -Aminoisobutyrate also arises
  by transamination of methylmalonate semialdehyde,
  a catabolite of L-valine .
• The initial reaction of -alanine catabolism is
  transamination to malonate semialdehyde.
  Subsequent transfer of coenzyme A from
  succinyl-CoA forms malonyl-CoA semialdehyde,
  which is then oxidized to malonyl-CoA and
  decarboxylated to the amphibolic intermediate
  acetyl-CoA.

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Analogous reactions characterize the catabolism
of -aminoisobutyrate. Transamination forms
methylmalonate semialdehyde, which is converted
to the amphibolic intermediate succinyl-CoA by
reactions 8V and 9V of. Disorders of -alanine
and -aminoisobutyrate metabolism arise from
defects in enzymes of the pyrimidine catabolic
pathway. Principal among these
are disorders that result from a total or partial
deficiency of dihydropyrimidine dehydrogenase.
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• 2) beta-Alanyl Dipeptides
• The -alanyl dipeptides carnosine and anserine
  (N -methylcarnosine) activate myosin ATPase,
  chelate copper, and enhance copper uptake.
  -Alanyl-imidazole buffers the pH of anaerobically
  contracting skeletal muscle.
• Biosynthesis of carnosine is catalyzed by
  carnosine synthetase in a two-stage reaction that
  involves initial formation of an enzyme-bound
  acyl-adenylate of -alanine and subsequent
  transfer of the -alanyl moiety to L-histidine.

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• Hydrolysis of carnosine to -alanine and
  L -histidine is catalyzed by carnosinase. The
  heritable disorder carnosinase deficiency is
  characterized by carnosinuria.
• Homocarnosine, present in human brain at higher
  levels than carnosine, is synthesized in brain
  tissue by carnosine synthetase. Serum carnosinase
  does not hydrolyze homocarnosine.
  Homocarnosinosis, a rare genetic disorder, is
  associated with progressive spastic paraplegia
  and mental retardation.

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• 3) gama-Aminobutyrate
• gama-Aminobutyrate (GABA) functions in brain
  tissue as an inhibitory neurotransmitter by
  altering transmembrane potential differences.
• GABA is formed by decarboxylation of glutamate
  by L -glutamate decarboxylase. Transamination of
  -aminobutyrate forms succinate semialdehyde,
  which can be reduced to -hydroxybutyrate by
  L -lactate dehydrogenase, or be oxidized to
  succinate and thence via the citric acid cycle to
  CO2 and H2O.

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• A rare genetic disorder of GABA metabolism
  involves a defective GABA aminotransferase, an
  enzyme that participates in the catabolism of
  GABA subsequent to its postsynaptic release in
  brain tissue.
• Defects in succinic semialdehyde dehydrogenase
  are responsible for another rare metabolic
  disorder of -aminobutyrate catabolism
  characterized by 4-hydroxybutyric aciduria.

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• 4) amino levulinic acid (ALA)
• ALA is intermediate in the synthesis of porphyrin
  (finally heme)

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5) Taurine

• Taurine (2-aminoethylsulphonic acid) is a
  non-protein
• aminoacid present in almost all animal tissues
  and
• the most abundant free intracellular aminoacid in
• human cells.
• In humans, it is considered to be a
  semi-essential
• aminoacid since it can be synthesized from
  other sulfonic aminoacids such as methionine and
  cysteine, in
• the presence of vitamin B6,2,3 but endogenous
  production is insufficient, so that it needs to
  be provided through diet.
• Taurine is found in association with bile
  acids.

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• Biological effects of taurine in the context of
  diabetes
• Biological EffectMechanismof Taurine
• Antioxidant action By inhibiting ROS generation
  at mitochondria Osmoregulation By counteracting
  osmotic inbalance through cellular membrane due
• to hyperglycaemia
• Antiinflammatory effects By interfering the
  formation of inflammatory mediators Glucose
  Homeostasis By interfering the insulin signalling
  pathway acting upon UCP2 protein

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• Estimation of NPAAs
• 1) The aim of our study was to analyze NPAAs
  and D-AA s in biosamples by means of capillary
  electrochromatogrphy (CEC) using a chiral
  practicle- loded monolithiac column with
  flurrosense detection for high sensitivity.
• 2) capillary electrophoresis
• 3) High perfromance liquid chromatography(HPLC)
• 4) laser-induced flurosecne (LIF)
• 5) scanning electro microscopy (SEM)

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• Scanning electro micrograph of CEC capillary
  coulmn

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Location of the regions of ordered secondary
structures for b-residues in fqy space. The
a-helix and b-sheet are the classical structures
for poly a-amino acids. b-residues occurring in
the appropriate shaded region can be
accommodated without disruption of secondary
structures. The 12-helix and 14-helix are
well characterised secondary structures for poly
b-peptides.
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THANK Q