Amino acids

DR/SA;;Y C.SUBA

Contents
1. 2. 3. 4. 5. Introduction Structures of Amino acids Metabolism of Amino acids Aminocidopathies Amino Acid Analysis

Introduction
i. Building blocks of proteins ii. Growth, repair and maintenance of cells

Peptide bond

Amino Acid

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 .

: Nitrated tyrosine (a) and tryptophane (b)

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)

Chemical Properties
MILLONS TEST-This test, also known as Coles 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.

 PAULYS 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.

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, -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 ketoglutarate produced glutamate. In this reaction, glutamate is the major donor of amino group while -ketoglutarate is the major acceptor of amino group: NH4+ + -ketoglutarate+ NADPH + H+ + NADP+ + H2O glutamate

Then glutamate is converted to glutamine which is catalyzed by glutamine synthetase: NH4+ + glutamate + ATP Pi glutamine + ADP+

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 -ketoglutarate as the precursor of glutamate. Aspartate Family: include asparagine, methionine, threonine, lysine and isoleucine with - 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-4phosphate 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.

 Glutamate and Glutamine. Glutamate (glutamic acid) is synthesized from the transamination of -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 3phosphopyruvate. 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)

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------ -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, 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
Lysine leucine

Glycine Serine Valine Histidine Arginine Cysteine Proline Alanine Glutamate Glutamine Aspartate Asparagine Methionine

Both glucogenic and ketogenic Isoleucine Threonine Phenylalanine Tyrosine Tryptophan

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. Chains of amino acids are also used to synthesize small peptide hormones such asThyrotropinReleasing 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.

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

Aminoacidopathies
Class of inherited errors of metabolism Enzymes defects that inhibits the bodys ability to metabolize certain amino acids
i. ii. iii. iv. v. PKU Tyrosinemia Alkaptonuria MSUD Isovaleric Acidemia
vi. vii. viii. ix.

Homocystinuria Citrullinemia Arginosuccinic Aciduria Cystinuria

Aminoacidopathies

Metabolism of phenylalanine and tyrosine

Aminoacidopathies
i. Phenylketonuria
Absence of phenylalanine hydroxylase (PAH)
PAH

1.
2.

Laboratory Tests
Guthrie test
Microfluorometric assay

Aminoacidopathies
i. Phenylketonuria

1.

Laboratory Tests
Guthrie test Semi quantitative bacterial inhibition assay Uses phenylalanine to facilitate bacterial growth (B. subtilis and -2-thienylalanine).

Aminoacidopathies
i. Phenylketonuria

2.

Laboratory Tests
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

Phenylalanine hydroxylase

Phenylketonuria
Tyrosine aminotransferase

Tyrosinemia II

Tyrosinemia III
4-hydroxyphenylpyruvate dioxygenase

Alkaptonuria

Homogentisate oxidase

Fumarylacetoacetate hydrolase

Tyrosinemia I

Aminoacidopathies
vi. Maple Syrup Urine Disease
branched-chain -ketoacid decarboxylase

Aminoacidopathies
vi. Maple Syrup Urine Disease

1.

Laboratory Tests
Modified Guthrie test uses branched chain -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 - synthetase

Aminoacidopathies
viii.Homocystinuria

1.

Laboratory Tests
Modified Guthrie test Uses methionine to facilitate bacterial growth (containing B. subtilis and L-methionine sulfoximime).

Aminoacidopathies
ix. Citrullinemia
i.

Type I citrullinemia
Lack of arginonosuccinic acid synthetase (ASAS)

Aminoacidopathies
ix. Citrullinemia
i.

Type II citrullinemia
Mutation of the gene that encodes for protein citrin

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

Aminoacidopathies
ix. Citrullinemia x. 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 bodys ability to metabolize certain amino acids
i. ii. iii. iv. v. PKU Tyrosinemia Alkaptonuria MSUD Isovaleric Acidemia
vi. vii. viii. ix.

Homocystinuria Citrullinemia Arginosuccinic Aciduria Cystinuria

Disease
PKU

Enzyme deficiency
Phenylalanine hyroxylase Tyrosine aminotranferase 4-Hydroxyphenylpyruvate oxidase

Amino acid increased

Phenylalanine Tyrosine p-Hydroxyphenylpyruvic acid

Tyrosinemia I
Tyrosinemia II Tyrosinemia III

Fumarylacetoacetate hydrolase Fumarylacetoacetate

Alkaptonuria
MSUD

Homogentisate oxidase
Branched-chain -ketoacid decarboxylase

Homogentisic acid (HGA)

Leucine, Isoleucine, Valine Leucine, Isovaleric acid Methionine, homocysteine Citrulline, ammonia

Isovaleric acidemia Isovaleryl-CoA dehydrogenase Homocystinuria Citrullinemia Cystathionine- synthetase Arginosuccinic acid synthetase Argininosuccinic acid lyase Defective AA transport system

Arginosuccinic aciduria
Cystinuria

Argininosuccinic acid, Citrulline, ammonia

Cystine ppt

Aminoacidopathies
Diagnosis
i. ii. iii. iv. v. 6-8 hours fasting Collected in heparin tube with the plasma Deproteinization performed within 30 minutes Screening by TLC stained with ninhydrin (blue) Separated and quantitated by Ion exchange chromatography and HPLC reversed-phase system or capillary electrophoresis