non protein amino acids

NON PROTEIN

AMINOACIDS

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

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.

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

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

1) 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

2) NON ALPHA amino acids:

a) beta –alanine

b) beta – aminoisobutyric acid

c) gama – aminobutyric acid(GABA)

d) aminolevulinic acid (ALA)

e) taurine

Alpha - aminoacids1) 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

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

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.

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 1–2 g of citrulline are

excreted daily.

4) Thyrosine and triodothyroxine

: Tyrosine forms norepinephrine and

epinephrine, and following iodination the thyroid hormones triiodothyronine and thyroxine.

Use of measurement of blood thyroxineor 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)

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 10.3–21.9 pmol/L)

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

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 pyridoxalphosphate (vitamin B6)-dependent cystathionineb-synthase increase plasma methionine levels.

Homocysteine is transformed into methionine via remethylation. This occurs through methioninesynthase, a reaction requiring methylcobalaminand folic acid.

Deficiencies in these enzymes or lack of cofactors is associated with decreased or normal methioninelevels. In an alternative pathway, homocysteinecan be remethylated by betaine:homocysteinemethyl transferase

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.

Total plasma homocysteine is also extremely

elevated (usually >100 M). Treatment consists

of a special diet restricted in protein and

methionine and supplemented with cystine.

In approximately half of patients, oral

pyridoxine (25–500 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.

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 hydroxylatetyrosine 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-methyltransferase 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

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 dihydroxyphenylalanine or DOPA.

The phenyl ring with two adjacent OH groups is a

catechol, andhence dopamine, norepinephrine, and

epinephrine are called catecholamines.

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.

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.

Normal values :

Creatinine : 200 mol/L (44–80 mol/L)

Female -- 44–80 mol/L 0.5–0.9

ng/mL

male -- 53–106 mol/L 0.6–1.2

ng/mL

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 purinesynthesis have been used in cancer chemotherapy.

Inhibitory compounds and the reactions they inhibit include azaserine, diazanorleucine, 6-mercaptopurine , and mycophenolic acid .

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 amphibolicintermediate acetyl-CoA.

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.

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.

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.

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.

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.

4) amino levulinic acid (ALA) :

ALA is intermediate in the synthesis of

porphyrin (finally heme)

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.

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

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)

Scanning electro micrograph of CEC capillary coulmn

Location of the regions of ordered secondary structures for b-residues in

f–q–y 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

THANK Q