Amino acidsDR/SA;;Y C.SUBA

Contents

1. Introduction2. Structures of Amino acids3. Metabolism of Amino acids4. Aminocidopathies5. Amino Acid Analysis

Introduction

i. Building blocks of proteinsii. Growth, repair and maintenance of cells

Amino Acid

Peptide bond

Structures of 20 Amino Acids

PHYSICAL PROPERTIES OF AMINO ACIDS

White, crystalline

Soluble in cold water, except cystine and tyrosine which are soluble in hot water. However, its solubility in alcohol and ether except proline and hydroxyproline because of its nature.

All amino acids are precipitated by alcohols except proline but not by (NH4)2SO4 or NaCl.

Some are sweet like glycine, alanine, serine, and proline;leucine is tasteless: arginine is bitter.

Chemical Properties

• Ninhydrin Reaction Ninhydrin( Triketohydrinthinhydrtae) is reduced to hydrindantin. The amino acid is dismuted to an aldehyde, ammnia, and carbon dioxide. The formation of colored chromogen is made by the reaction of Hydrindantin with ammonia and another molecule of ninhydrin .

Chemical Properties

• XANTHOPROTEIC TEST- Using 65% nitric acid the aromatic rings of amino acids like tyrosine and tryptophan are nitrated. The nitro derivate shows an intensely yellow color. Because nearly all proteins contain aromatics it is taken as a protein-test either. : Nitrated tyrosine (a) and tryptophane (b)

: Nitrated tyrosine (a) and tryptophane (b)

Chemical Properties

• MILLON’S TEST-This test, also known as Cole’s test, is answered by compounds containing hydroxphenyl group. Tyrosine as shown in figure 7.43 both in free and bound forms react with Sodium nitrite under acid condition and is converted to nitrous acids. The end product is probably due to the formation of mercury phenolate with nitrated phenyl group

Chemical Properties

• SAKAGUCHI TEST• Arginine, the only amino acid with a guanidium group that gives a red

olour with napthol and Sodium hypobromitein the presence of an oxidizing agent such as Cl2 /Br2 .

Chemical Properties• SULFUR TEST

• By boiling with NaOH, S in the amino acid is converted into Na2S, which then precipitates as blackPbS with the addition of lead acetate.When proteins containing cysteine or cystine residues are boiled under strong alkali organic sulfur,it is converted to sulfide. Addition of lead acetate causes precipitation of insoluble black lead sulfide.

• PAULY’S TEST- The test is for imidazole group of histidine and hydroxyphenyl group of tyrosine. Diazobenzene sulfuric acid formed in the reaction condenses with histidine to form a cherry red color, diazotized product under alkaline condition. With tyrosine, an orange-red colored product is obtained.

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STEREOCHEMISTRY OF AMINO ACIDS

STEREOCHEMISTRY OF AMINO ACIDS

• R and S Forms:If the priorities of these other groups go in a clockwise rotation, the chiralty is “R’. If the priorities of these other groups go counterclockwise, the chiralty is “S”. (Note that this assignment has nothing to do with optical activity, and is not using L-glyceraldehyde as a reference molecule)

• Cis and Trans

CLASSES OF AMINO ACIDS

• BASED ON POLARITY OF SIDE CHAIN• 1. Group.1 Amino acid with non-polar hydrophobic side chain.•  This group-1 amino acids are glycine, alanine, valine, leucine, isoleucine, proline,

phenylalanine, methionine and tryptophan. They have carbon-hydrogen containing aliphatic or aromatic groups and are non-polar.

• 2. Group II Amino acids with Polar, Uncharged Side Chains at• physiologicalpH.• The amino acids of the group- II are serine, cysteine, threonine, tyrosine,

asparagine, glutamine and histidine. All of these amino acids have at least, heteroatom (N, O and S) with electron pairs available for hydrogen bonding to water and other molecules. The most fascinating member of this group is cysteine with an -SH (sulfhydryl group on the side chain, which reacts under oxidizing conditions with -SH group of another cysteine forming disulfide bond.

CLASSES OF AMINO ACIDS

• BASED ON POLARITY OF SIDE CHAIN

• 3. Group III Amino Acids with Polar, Charged side chains at physiologicalpH.

•• The amino acids of group III are aspartate,

glutamate,glysine, and arginine.At physiologic pH, the side chains ofaspartate and glutamate dissociate protons to form carboxylate anions.

CLASSES OF AMINO ACIDS• BASED ON THE NUMBER OF AMINO ACIDS AND CARBOXYLIC • ACIDS• • 1. Aliphatic, monoamino monocarboxylic acids (neutral)• • Examples of these groups are listed as follows:• Glycine - aminoacetic• Alanine - alphaamino propionic acid• Valine - alpha amino gamma methylbutyric acid• Leucine - alpha amino gamma methylvaleric acid• Isoleucine - alpha amino betamethylvaleric acid• Noneucine - alpha amino caproic acid• Serine - alpha amino beta hydroxybutyric acid• Threonine - alpha amino betahydroxybutyric acid• Cysteine - alpha amino betathiopropionic acid• Cystine - di (alpha amino beta thiopropionic acid)• Methionine - alpha amino gamma methythiolbutyric acid•

CLASSES OF AMINO ACIDS• BASED ON THE NUMBER OF AMINO ACIDS AND CARBOXYLIC • ACIDS

•  2. Monoaminodicarboxylic acids (acidic amino acids)• Aspartic acid - aminosuccinic acid

• Glutamic acid - alpha amino glutaric acid•  • 3. Aromatic Amino Acids (contain aromatic ring)•   Phenylalanine - alpha amino beta phenylpropionic acid• Tyrosine - alpha amino beta parahydroxyphenyl• propionic acid•  4.Heterocyclic Amino Acids•   Tryptophan (also aromatic) - alpha amino beta indole– propionic acid• Histidine (also aromatic) - alpha amino beta imidazole – propionic acid• Proline (non-aromatic) alpha pyrolidine carboxylic acid• Hydroxyproline (non-aromatic) - gamma hydroxyl alpha pyrrolidine carboxylic acid•  • 5.Diamino Monocarboxylic acid (basic amino acids)•

• Arginine - alpha amino lambda guanidinovaleric acid• Lysine - alpha, epsilon diaminocaproic acid•  

CLASSES OF AMINO ACIDS• BASED ON CHEMICAL COMPOSITION• • 1. According to Structure• Aliphatic amino acid - carbon atoms are arranged in a straight chain

(lysine, arginine, glutamic acid, aspargine, glutamine)• Branched amino acid - branching of side chain. (leucine, isoleucine, valine)• Aromatic amino acid - contains the benzene ring (phenylalanine, tyrosine)• Heterocyclic amino acid - cyclic side chain containing C atoms and other

elements (tryptophan, histidine, proline, hydroxyproline)• Sulfur-containing – contains S (cysteine, cystine)• Hydroxy-containing - contains -OH group (threonine, serine)• Iodine- containing (thyroxine)•

CLASSES OF AMINO ACIDS

• BASED ON CHEMICAL COMPOSITION• 2. According to Acidity• Acidic amino acids - with more -COOH than -NH2

group (aspartic acid, glutamic acid)• Basic amino acids - with more -NH2 than -COOH

group ( lysine, arginine)• Neutral amino acids - with one -COOH and one

NH2 (glycine, alanine)•

CLASSES OF AMINO ACIDS• BASED ON CHEMICAL COMPOSITION• 3.According to Interaction of Side Chains• • Non-polar or hydrophobic - R group forms hydrophobic interactions which can

impart insolubility of proteins in water (alanine, valine, leucine)• • Polar or Hydrophilic Group (Uncharged) - R group that is polar• 1. -SH (cysteine)• 2 - NH2 (lysine)• 3 -OH (serine, threonine, tyrosine)• • Aromatic Side Chains - show stacking interactions and pi-pi complexion• Ionic or Charged Side Chains - ionized acidic amino acids (aspartic acid, glutamic

acid), and ionized basic amino acids (lysine, arginine, histidine).•

CLASSES OF AMINO ACIDS• BASED ON NUTRITION VALUE• • 1. Essential amino acids - amino acids that cannot be synthesized in • sufficient quantities and must be supplied in the diet of humans.• • These amino acids are arginine, histidine, isoleucine, leucine, lysine, • methionine, phe, thronine, tryptophan and valine.• • 2.Nonessential amino acids - amino acids that are synthesized by

humans.• • These amino acids include alanine, asparagines, aspartate, cysteine, • glutamate, glutamine, glycine, proline, serine and tyrosine.

Additional Information

i. Nutritionally essential amino acids is supplied by diet (e.g. histidine, lysine, etc.)

ii. Some amino are produced in the body (e.g. cysteine, glutamine, etc.)

Nitrogen cycle •

• Processes involved in nitrogen cycle:•  • Nitrogen Fixation. Molecular nitrogen

(N2) is inert and does not react to form compounds. Substantial amounts of energy are needed to break it apart so that its atoms can combine with other atoms.

• The process of transforming nitrogen to a usable form is known as nitrogen fixation.

• Three processes are involved in the nitrogen fixation in the atmosphere: atmospheric fixation by lightning, biological fixation by certain microorganism and industrial fixation.

Nitrogen cycle • Nitrification. It involves the

oxidation of the ammonium compounds in dead organic material into nitrites and nitrates by nitrifying bacteria.

• Nitrosomonas bacteria can oxidize NH3 to nitrites (NO2

−) while Nitrobacter bacteria can oxidize the nitrites to nitrates (NO3

−)

• .These bacteria live in the roots of legume plants such as peanuts, beans, and peas.

Nitrogen cycle

• Denitrification. It is the process of reducing nitrates to nitrogen gas which replenish the nitrogen in the atmosphere

Overview of amino acid metabolism

• Degradation of amino acid. The carbon skeleton from the degradation of amino acids will give rise to metabolic intermediates such as pyruvate, acetyl CoA, α-ketoglutarate, succinyl CoA, fumarate and oxaloacetate.

• These metabolites can enter the citric acid cycle or can be used in gluconeogenesis.

• Among the metabolites, oxaloacetate is considered as key intermediate because of its dual role in the citric acid cycle and gluconeogenesis. (Figure 14.2)

• The carbon skeletons of the metabolites that enter the citric acid cycle will be completely oxidized into CO2 or diverted into gluconeogensis or ketogenesis.

Overview of amino acid metabolism

• • Transamination reactions. The nitrogen portion of

amino acids is involved in the transamination reactions. Excess nitrogen is excreted in three forms: ammonia (ammonium ion), urea and uric acid. The reaction between ammonia and α-ketoglutarate produced glutamate. In this reaction, glutamate is the major donor of amino group while α-ketoglutarate is the major acceptor of amino group:

• • NH4

+ + α-ketoglutarate+ NADPH + H+ glutamate + NADP+ + H2O

• • Then glutamate is converted to glutamine which is

catalyzed by glutamine synthetase:• • NH4

+ + glutamate + ATP glutamine + ADP+ Pi

• •

Families of Amino acids• Amino acids can be group by families

since they have common precursor in the biosynthetic pathways:

• • Glutamate Family: include glutamine,

proline and arginine with α-ketoglutarate as the precursor of glutamate.

• Aspartate Family: include asparagine, methionine, threonine, lysine and isoleucine with α- oxaloacetate as theprecursorof aspartate.

• Serine Family: include cysteine and glycine 3- phosphoglycerate as the precursor of serine.

• Pyruvate Family: include valine, alanine and leucine with

• pyruvate as the precursor.

• Aromatic Family: include phenylalanine and tyrosine with

• phosphoenolpyruvate as the precursor, and erythrose-4-phosphate as the precursor of tyrosine and tryptophan .

• Histidine Family: include histidine with ribose-5-phosphate as the precursor.

Non-essentia Amino acid Biosynthesis

• Alanine.It can be synthesized by transamination of pyruvate by enzyme alanine transaminase (ALT):

Non-essentia Amino acid Biosynthesis

• Asparagine. Asparagine is derived from aspartate. The precursor to aspartate is oxaloacetate:

Non-essentia Amino acid Biosynthesis

• Transamination wherein the amino group from glutamate is transferred to oxaloacetate which is catalyzed by aspartate transaminase (AST), (2) deamination of asparagine which is catalyzed by asparaginase:

Non-essentia Amino acid Biosynthesis

• Cysteine.Cysteine, a sulfur containing amino acid, is synthesized from homocysteine which is derived from metabolism of another sulfur containing amino acid, methionine. The homocysteine condenses with serine to form cystathionine, which is deaminated and hydrolyzed to form cysteine and alpha-ketobutyrate.

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• Glutamate and Glutamine. Glutamate (glutamic acid) is synthesized from the transamination of α-ketoglutarate which is catalyzed by glutamate dehydrogenase. Glutamine is formed when a second ammonium ion is incorporated into glutamate by the action of the enzyme glutamine synthetase:

Non-essentia Amino acid Biosynthesis

• Proline and Arginine. Glutamate is the precursor of proline and arginine:

Non-essentia Amino acid Biosynthesis

•Serine and Glycine.It is derived from 3-phosphoglycerate which is an intermediate metabolite from glycolysis. The 3-phosphoglycerate is converted to a keto acid which is the 3-phosphopyruvate. Then the amino group from glutamate is transferred to 3-phosphopyruvate to form 3-phosphoserine which is converted to serine catalysed by the enzyme phosphoserine

phosphatase.

Serine is the precursor of glycine:

Amino Acid Pool• When there is high level of amino acids

in the body, the liver stores them until needed.

• Amino acids are withdrawn from the amino acid pool for various purposes:

• (1) synthesis of glucose, glycogen, and lipid;

• (2) synthesis of non-protein compounds, heme and heterocyclic amines;

• (3) synthesis of other amino acids; • (4) synthesis of hormones, enzymes,

and tissue and plasma proteins.Other amino acids are oxidized to CO2 and H2O to generate ATP (Figure 14.10)

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Catabolism of Amino acids

• 1 Ala, Cysteine,Glycine, Serine and Tryptophan --------------- Pyruvate

• 2, Leu, Lys, Phe, Tryp, Tyr---------- Acetyl CoA• 3. Asp and Aspn ======= oxaloacetate• 4. Phe and Tyr --------------- Fumarate• 5. Ile, Leu, and Tryp---------- Acetyl CoA• 6. Arg, Gln,Glu, His, Pro------ α-Ketoglutarate• 7. Ile, Met, Thr, and Val --------Succinyl CoA

Fate of Carbon Skeleton of Amino Acids

• The breakdown of carbon skeleton of amino acids follows different pathways depending on the type of amino acids:

• Glucogenic amino acids are those that produce pyruvate, α-ketoglutarate or oxaloacetate which can be converted into glucose through gluconeogenesis.

• Ketogenic amino acids give rise to acetylCoA or acetoacetylCoA which can be converted into ketone bodies.

• Table 14.1 shows the amino acids that are considered glucogenic, ketogenic or both.

• •

Fate of Carbon Skeleton of Amino Acids

Glucogenic Ketogenic Both glucogenic and ketogenic

GlycineSerineValineHistidineArginineCysteineProlineAlanineGlutamate GlutamineAspartateAsparagineMethionine

Lysineleucine

IsoleucineThreoninePhenylalanineTyrosineTryptophan

Regulation of Amino acids • 1. Amino acids like tyrosine,

phenylalanine and tryptophan are used to synthesize hormones such as catecholamines and thyroxine.

• Catecholamines such as epinephrine (adrenaline), norepinephrine (noradrenaline) and dopamine which are produced from phenylalanine and tyrosine are released from the adrenal medulla of the adrenal glands as part of the fight-or-flight response.

• Thyroxine is a hormone synthesized from tyrosine which is secreted by the thyroid gland that regulate metabolism.

• Chains of amino acids are also used to synthesize small peptide hormones such asThyrotropin-Releasing Hormone(TRH) and vasopressin.

• TRH is a tripeptidal hormone that stimulates the release of TSH (thyroid-stimulating hormone) and prolactin from the anterior pituitary.

• Vasopressin is a nine amino acid peptide secreted from the posterior pituitary which constricts blood vessels, raises blood pressure, stimulates intestinal motility, and reduces the excretion of urine.

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Aminoacidopathies

Class of inherited errors of metabolism Enzymes defects that inhibits the body’s ability to

metabolize certain amino acidsi. PKUii. Tyrosinemiaiii. Alkaptonuriaiv. MSUDv. Isovaleric Acidemia

vi. Homocystinuriavii. Citrullinemiaviii. Arginosuccinic Aciduriaix. Cystinuria

Aminoacidopathies

Metabolism of phenylalanine and tyrosine

Aminoacidopathies

i. Phenylketonuria Absence of phenylalanine hydroxylase (PAH)

Laboratory Tests1. Guthrie test

2. Microfluorometric assay

PAH

Aminoacidopathies

i. Phenylketonuria Laboratory Tests

1. Guthrie test Semi quantitative bacterial inhibition assay Uses phenylalanine to facilitate bacterial growth

(B. subtilis and β-2-thienylalanine).

Aminoacidopathies

i. Phenylketonuria Laboratory Tests

2. Microfluorometric assay Direct quantitative measurement Phenylalanine – ninhydrin – copper fluorescence

in the presence of dipeptide (360-530 nm)

Aminoacidopathies

ii. Type II Tyrosinemia ↓ Tyrosine aminotransferase

iii. Type III Tyrosinemia ↓ 4-hydroxyphenylpyruvate dioxygenase

Aminoacidopathies

v. Alkaptonuria Lack of homogentisate oxidase

vi. Type I Tyrosinemia ↓ fumarylacetoacetate hydrolase

Phenylketonuria

Tyrosinemia II

Tyrosinemia III

Alkaptonuria

Tyrosinemia I

Phenylalanine hydroxylase

Tyrosine aminotransferase

4-hydroxyphenylpyruvate dioxygenase

Homogentisate oxidase

Fumarylacetoacetate hydrolase

Aminoacidopathies

vi. Maple Syrup Urine Disease ↓ branched-chain α-ketoacid decarboxylase

Aminoacidopathies

vi. Maple Syrup Urine Disease Laboratory Tests

1. Modified Guthrie test uses branched chain α-ketoacid to facilitate bacterial

growth (containing B. subtilis and 4-azaleucine).

Aminoacidopathies

vii. Isovaleric Acidemia Deficiency of isovaleryl-CoA dehydrogenase

Aminoacidopathies

viii.Homocystinuria Lack of cystathionine β- synthetase

Aminoacidopathies

viii.Homocystinuria Laboratory Tests

1. Modified Guthrie test Uses methionine to facilitate bacterial growth

(containing B. subtilis and L-methionine sulfoximime).

Aminoacidopathies

ix. Citrullinemiai. Type I citrullinemia

Lack of arginonosuccinic acid synthetase (ASAS)

Aminoacidopathies

ix. Citrullinemiai. Type II citrullinemia

Mutation of the gene that encodes for protein citrin

Aminoacidopathies

x. Argininosuccinic aciduria Lack of argininosuccinic acid lyase (ASL)

Aminoacidopathies

ix. Citrullinemiax. Argininosuccinic aciduria

Aminoacidopathies

xi. Cystinuria Defect in amino acid transport system by

inadequate reabsorption of cystine in the kidneys

Aminoacidopathies

Class of inherited errors of metabolism Enzymes defects that inhibits the body’s ability to

metabolize certain amino acidsi. PKUii. Tyrosinemiaiii. Alkaptonuriaiv. MSUDv. Isovaleric Acidemia

vi. Homocystinuriavii. Citrullinemiaviii. Arginosuccinic Aciduriaix. Cystinuria

Disease Enzyme deficiency Amino acid increasedPKU Phenylalanine hyroxylase PhenylalanineTyrosinemia I Fumarylacetoacetate hydrolase FumarylacetoacetateTyrosinemia II Tyrosine aminotranferase Tyrosine

Tyrosinemia III 4-Hydroxyphenylpyruvate oxidase

p-Hydroxyphenylpyruvic acid

Alkaptonuria Homogentisate oxidase Homogentisic acid (HGA)

MSUD Branched-chain α-ketoacid decarboxylase

Leucine, Isoleucine, Valine

Isovaleric acidemia Isovaleryl-CoA dehydrogenase Leucine, Isovaleric acid

Homocystinuria Cystathionine-β synthetase Methionine, homocysteine

Citrullinemia Arginosuccinic acid synthetase Citrulline, ammoniaArginosuccinic aciduria Argininosuccinic acid lyase Argininosuccinic acid,

Citrulline, ammoniaCystinuria Defective AA transport system Cystine ppt

Aminoacidopathies

Diagnosisi. 6-8 hours fastingii. Collected in heparin tube with the plasmaiii. Deproteinization performed within 30 minutesiv. Screening by TLC stained with ninhydrin (blue)v. Separated and quantitated by Ion exchange

chromatography and HPLC reversed-phase system or capillary electrophoresis

End of Part I