Chemistry of Amino Acids

Amino acids are the units of proteins, and understanding its chemistry and the the properties assists in understanding the functions of proteins. This gives in an idea to why a certain protein behaves in a certain way.

1. Chemistry of Amino Acids(A Ᾱ s)

2. Amino Acids What are amino acids ?? Amino acids are organic compounds. Organic compounds are the substances that are made up of Hydrocarbon (Hydrogen and Carbon) and its derivatives.

3. Contents Definition History General Structure Classification Properties Physical Isomerism Optical activity and stereoisomerism Spectroscopic property of amino acid Iso -electric point Chemical Reactions Color reactions Biosynthesis Catabolism Separation and analysis of amino acid mixture Functions References

4. Definition The basic monomeric structural unit of proteins organic in nature whose constituent are two functional groups, an amino group (-NH 2 ) which is basic in nature and a carboxylic group (-COOH) which is acidic in nature are called as amino acids.

5. History

6. General Structure of an Amino Acid Amino group Hydrogen Side-chain Carboxylic group α Carbon

7. Classification Based on structure Based on metabolic fate Based on side chain characters Based on nutritional requirement

8. A. Based on structure Aliphatic amino acids Monoamino monocarboxylic acid Simple AĀs - Gly , Ala. Branched chain AĀs – Val, Ile, Leu . Hydroxy AĀs – Ser, Thr . Sulphur containing AĀs – Cys , Met. AĀs with amide group – Asn , Gln . Monoamino dicarboxylic acid – Asp, Glu . Dibasic monocarboxylic acid – Lys, Arg. Aromatic AĀs – Phe , Tyr. Heterocyclic AĀs – Trp , His. Imino acids – Pro, hydroxyproline Derived AĀs Derieved AĀs found in protein – hydroxyproline , hydroxylysine Derieved amino acids not seen in proteins – ornithine , citrulline , homocysteine . Non alpha AĀs – GABA.

9. B. Based on metabolic fate Purely Ketogenic Leu . Sometimes Lys is also considered Ketogenic & Glucogenic Lys, Ile, Phe , Tyr, Trp. Purely Glucogenic All the remaining 14.

10. C. Based on side chain Characters Hydrophilic or polar AĀs Charged hydrophilic - vely charged – Asp, Glu + vely charged – Lys, Arg , His Uncharged hydrophilic Thr , Ser with OH side chain Asn , Gln with amide side chain Cys with SH side chain Gly with hydrogen also considered

11. 2. Hydrophobic or non polar AĀs Hydrophobic with aliphatic side chain Val, Ala , Ile, Leu Hydrophobic with aromatic side chain Phe , Tyr, Trp C. Based on side chain Characters

12. Based on nutritional requirement Essential/Indispensable Ile, Leu , Thr , Lys, Met, Phy , Trp , Val. Semi-essential His, Arg Non-essential Remaining 10

13. alanine valine leucine isoleucine proline phenylalanine tyrosine tryptophan methionine serine threonine

14. cysteine asparagine glutamine aspartate glutamate lysine histidine

15. Properties Physical Colourless, crystalline substances Taste Sweet: Gly , Ala, Val, Ser, Trp , His & Pro. Tasteless: Leu . Bitter: Ile & Arg. Artificial sweetener: Aspartame contains Asp & Phy . Melting point: above 200˚C. High dielectric constant Solubility Water: mostly all. Alcohol(polar solvent): mostly all. Benzene(non-polar solvent): none. Tyr is soluble in hot water. defined as the product of magnitude of charge & distance of separation between the charges.

16. Asymmetric carbon Chirality Enantiomer Stereoisomerism Plane polarised light Zwitterion

17. Isomerism Of the standard α- AĀs, all but Gly can exist in either of two optical isomers, called L or D AĀs, which are mirror images of each other. L-amino acids represent all of the AĀs found in proteins D- AĀs are found in some proteins as in exotic sea-dwelling organisms such as cone snails, are also abundant components of the peptidoglycan cell walls of bacteria, D-serine may act as a neurotransmitter in the brain. The isomers are of two types Optical isomer Stereoisomer

18. Optical activity and stereochemistry of amino acids With the exception of Gly , all the 19 other common AĀs have a uniquely different functional group on the central tetrahedral alpha carbon( α -C) The α -C is termed " chiral " to indicate there are four different constituents and that the α -C is asymmetric Since the α -C is asymmetric there exists two possible, non- superimposable , mirror images of the amino acids

19. The D, L system When looking down the H-C a bond towards the α -C there is a mnemonic to identify the L- enantiomer of Aās L- enantiomer , CORN. CONR (a silly, meaningless word) for the D- enantiomer .

20. Stereoisomerism of Amino Acids Levo-rotatory Dextro- rotatory

21. Enantiomeric molecules have an optical property known as optical activity - the ability to rotate the plane of plane polarized light Clockwise: dextrorotatoy Counterclockwise: levorotatory All common AĀs are the L- enantiomer However, not all AĀs are Levorotatory, some are actually Dextrorotatory with regard to their optical activity To (attempt) to avoid confusion, the optical activities are given as (+) for dextrorotatory, and (-) for levorotatory L(+)- alanine L(-)-serine

22. Spectroscopic properties of AĀs This refers to the ability of AĀs to absorb or emit electromagnetic energy at different wavelengths (i.e. energies) No AĀs absorb light in the visible spectrum (i.e. they are "colorless"). All AĀs absorb in the IR (longer wavelengths, weaker energy than visible light) Some AĀs absorb in the UV spectrum (shorter wavelengths, higher energy than visible light) Absorption occurs as electrons rise to higher energy states Electrons in aromatic ring structures absorb in the UV. spectrum. Such structures comprise the side chains of Trp , Tyr & Phy .

23. Isoelectric point Isoelectric point ( pI ) is the pH at which a particular molecule or surface carries no net electrical charge or the negative(- ve ) and positive(+ ve ) charges are equal. Zwitterions contain both + ve and - ve charges depending on the functional group. Net charge on the molecule is affected by pH of their surrounding & can become more + vely or - vely charged d/t loss or gain of protons (H + ). Calculating pI values For an AĀs with only one amine and one carboxyl group, the pI can be calculated from the mean of the pKas of this molecule.

24. Glycine has two ionizable groups: a COOH group and NH 2 group, with pKa values of 2.34 and 9.6 respectively. pI =(2.34+9.6)/2 = 5.97

25. For simple AĀs such as Ala , the pI is an average of the pK a 's of the carboxyl (2.34) and ammonium (9.69) groups. Thus, the pI for Ala is calculated to be: (2.34 + 9.69)/2 = 6.02. In the case of Asp , the similar acids are the alpha-carboxyl function ( pK a = 2.1) and the side-chain carboxyl function ( pK a = 3.9), so pI = (2.1 + 3.9)/2 = 3.0. For Arg , the similar acids are the guanidinium species on the side-chain ( pK a = 12.5) and the alpha-ammonium function ( pK a = 9.0), so the calculated pI = (12.5 + 9.0)/2 = 10.75.

26. Chemical Reactions Reactions due to –COOH group Decarboxylation . Histidine Histamine +CO2 Tyrosine Tyramine + CO2 Lysine Cadavarine + CO2

27. 2. Formation of amides -COOH group of dicarboxylic AĀs can combine with ammonia to from the corresponding amide. Aspartic acid + NH3 Asaparagine 3. Reduction to amino alcohol Achieved in presence of lithium aluminium hydride 4. Formations of esters Can form esters with alcohols The –COOH group can be esterfied with alcohol Treatment with Na 2 CO 3 solution in cold releases free ester from ester hydrochloride.

28. Reactions due to –NH 2 group Transamination α amino group of AĀ can be transferred to α keto acid to form new AĀ and α keto acid. Important reaction in the synthesis of non essential AĀ. amino acid α - keto acid Amino acid α - keto acid

29. Fig: Oxidative deamination 2. Oxidative deamination α amino group is removed from the AĀ to form corresponding keto acid and ammonia. In body glutamic acid is the most common AĀ to undergo oxidative deamination

30. 3. Formation of carbamino compound Carbondioxide adds to α amino group of AĀs to form carbamino compound. Occurs at alkaline pH Serves as the mechanism for transport of CO2 from tissues to lungs by Hb .

31. Reactions due to side chain Transmethylation The methyl group of Met, after activation, may be transferred to an acceptor which becomes methylated Met + acceptor methylated acceptor + homocystiene

32. 2. Ester formation by OH group The hydroxy AĀs can form ester with phosphoric acid Ser & Thr are involved in the formation of phosphoprotein Similarly, these hydroxl group can form O- glycosidic bonds with carbohydrate residues to form glycoprotein. Reaction of the amide group The amide group of Gln & Asn can form N- glycosidic bond with with carbohydrate residues to form glycoproteins . Reaction of SH group Cys has a sulfhydryl (SH) group & it can form a disulphide bond (S-S)with another Cys residue The two Cys residue can connect two polypeptide chain by the formation of interchain disulphide bonds & links. The dimer formed by the two Cys residue is called cystine or Dicysteine .

33. Peptide bond formation Mistakenly called amino bond covalent bond formed b/w 2 molecules when the carboxyl group of one molecule reacts with the amino group of the another molecule, releasing a molecule of H 2 O. resulting CO-NH bond is the peptide bond, & resulting mol is an amide. peptide bond c/b broken down by hydrolysis peptide bonds formed within proteins have tendency to break when subjected to the +nce of H 2 O (metastable bonds) Peptide bond Elimination of water

34. E. Property due to both –COOH group and –NH 2 group Formation of chelated co-ordination complexes with certain heavy metals and other ions These include Cu ++ , Co ++ , Mn ++ & Ca ++ . O=C-O - O-C=O Ca ++ H 2 C-NH 2 NH 2 -CH 2 chelates are non ionic –used to remove calcium from bones and teeth

35. Color reaction of A Ᾱ s Ninhydrin reaction Adopted for qualitative & quantitative estimation Used for detection of A Ᾱ s in chromatography A Ᾱ s + 2 mols . Of Ninhydrin aldehyde with 1 C atom less + color complex Color complex: pink / purple / blue (c/a Ruhemann’s purple) Pro, hydroxy -Pro – yellow Gln , Asn (A Ᾱ s with amide group) – brown Xanthoproteic test Nitration rxn undergone by conc ņ HNO 3 Rings in Phe , Tyr & Trp End product yellow , intensified by alkaline medium Rxn causes the yellow stain in skin by HNO 3

36. Millon’s test Phenol group of tyrosine + HgSO4 H 2 SO 4 +NaNO 3 red color mercuric phenolate HgNO 3 /Hg(NO 3 ) 2 in HNO 3 c/b used Cl - interferes with the rxn so not suitable for tyrosine in urine samples Tapoica (deficient in Phe & Tyr) give – ve Millon’s & Xanthoproteic test Sakaguchi’s test for Arg : Arg + α - napthol + alk . Hypobromite bright red color This is d/t guanidium group

37. Aldehyde test for Trp : Hopkins-Cole test: Trp + glyoxylic acid (mix) Mix layered over H 2 SO 4 Violet ring at interface shows the + nce of indole ring Acree -Rosenheim rxn : Formaldehyde & HgSO 4 is used Ehrlich’s rxn : Para- dimethyl -amino- benzaldehyde & strong HCl Gives dark blue color Gelatin with limited Trp content do not give this test Pauly’s test for His/Tyr: Diazo -benzene sulfonic acid + imidazole group of His alkaline condition Diazotised product (cherry red color) Gives orange red color with phenol

38. Sulphur test for Cys : Cys boiled with strong alkali: o rganic S splits & forms Na 2 S, which on addition of Pb -acetate produces PbS ( black ppt ) Met doesn’t give this test as S in Met is in thio -ester linkage which is difficult to break

39. Biosynthesis Nitrogen is first assimilated into organic compounds in the form of glutamate, formed from alpha- ketoglutarate and ammonia in the mitochondrion. In order to form other AĀs, transaminase is used to move the amino group to another alpha- keto carboxylic acid. Eg , aspartate aminotransferase converts glutamate and oxaloacetate to alpha- ketoglutarate and aspartate.

40. Nonstandard AĀs are usually formed through modifications to standard AĀs. Eg , Homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S- adenosyl methionine . Hydroxyproline is made by a posttranslational modification of proline . Microorganisms and plants can synthesize many uncommon AĀs.

41. Catabolism AĀs can be: Glucogenic Ketogenic Both glucogenic and ketogenic

42. Degradation of AĀs often involve deamination by moving its amino group to alpha-ketoglutarate, forming glutamate. This process involves transaminases, often the same as those used in amination during synthesis. In many vertebrates, the amino group is then removed through the urea cycle and is excreted in the form of urea.

43. Separation & analysis of A Ᾱ s mixtures The 20 common AĀs differ from one another in several important ways. Here are just two: Mass Isoelectric point In looking at the isoelectric point of the different AĀs it seems that they will have different partial charges at a given pH. Eg , at pH 6.0 some will be - vely charged, and some + vely charged.

44. For those that are - vely charged, some will be slightly - ve , and others strongly - ve . Similarly, for those that are + vely charged, some will be slightly + ve , and others strongly + ve The charge differences of the AĀs means that they will have different affinities for other cationic or anionic charges

45. Ion Exchange Chromatography : Basis If the immobile surface was coated with anions, then the chromatography would be termed " cation exchange" chromatography (and cations would selectively bind and be removed from the solution flowing through).

46. Strength of binding can be affected by pH & salt concn of the buffer. The ionic species "stuck" to the column can be removed & collected by changing one of these conditions.

47. We could lower the pH of the buffer & protonate anions, this would eliminate their electrostatic attraction to the immobilized cation surface. Or, we could ↑se the salt concentration of the buffer, the anions in the salt would "compete off" bound anions on the cation surface.

48. Amino acid 3 & 1 letter abbvtn Mol. weight Molecular formula Melting point (◦C) Properties Leucine Leu /L 131 C6H13NO2 293 Production of GH Tyrosine Tyr/Y 181 C9H11NO3 290 ↑ses NTs, DOPA & NE Production of T3,T4,TSH Phenylalanine Phe/F 165 C9H11NO2 273 Formation of Ep Arginine Arg/R 174 C6H14N4O2 223 Precursor of nitric oxide Lysine Lys/K 146 C5H14N2O2 210 Treatment of HSV

49. Histidine His/H 155 C6H9N3O2 285 Released in allergic rxn Valine Val/V 117 C5H11NO2 315 Important in smooth nervous functioning Tryptophan Trp/W 182 C11h12N2O2 283 Production of serotonin Isoleucine Ile/I 131 C6H13NO2 287 Isomer of leucine Methionine Met/M 149 C5H11NO2S 284 Helps reduce the estrogen load prevents Ca

50. Amino acid 3 & 1 letter abbvtn Molecular weight Molecular formula Melting point Properties Threonine Thr /T 119 C4H9NO3 256 Elevates symptoms of multiple sclerosis Proline Pro/P 115 C5H9NO2 228 Diminishes arteriosclerosis Glutamine Gln /Q 146 C5H10N2O3 185 Protects GI lining Releases cortisol Neurotoxic effect & cancer prevention Cysteine Cys/C 121 C3H7NO2S 220 Produces GSH & Taurine Recovery of hair & nail ts . Aspartic acid Asp/D 133 C4H7NO4 270 Participates in ornithine cycle Imp in function of RNA/DNA

51. Glutamic acid Glu /E 147 C5H9NO4 205 Supports brain function Acts as NTs Serine Ser/S 105 C3H7NO3 222 Production of Abs Absorption of creatine Alanine Ala/A 63 C3H7NO2 315 Helps treat benign prostrate hyperplasia Glycine Gly/G 57 C2H5NO2 233 Protection against cancer by antioxidants ⅓ of collagen are glycine Asparagines Asn/N 132 C4H8N2O3 235 Biosynthesis of glycoproteins

52. Selenocysteine : The 21 st amino acid Abbreviated as Sec or U + nt in enzymes ( eg glutathione peroxidases, tetraiodothyronine 5' deiodinases , glycine reductases ) has a structure similar to that of cysteine, but with an atom of selenium taking the place of the usual sulfur, forming a selenol group. Proteins contain one or more selenocysteine residues are called selenoproteins encoded in a special way by UGA codon, which is normally a stop codon.

53. Pyrrolysine : abbreviated as Pyl or O is a naturally occurring, genetically coded amino acid used by some methanogenic archaea . It is similar to lysine, but with an added pyrroline ring linked to the end of the lysine side chain. Produced by a specific tRNA and aminoacyl tRNA synthetase , it forms part of an unusual genetic cod is considered the 22nd proteinogenic amino acid. encoded in mRNA by the UAG codo n

54. Functions Over 300 AĀs + nt & only 22 found in human body. Tyr forms hormones such as T 3 , T 4 , TSH , Ep, NE & pigment melanin Trp can synthesise Niacin Gly , Arg & Met can synthesise creatine Gly & Cys helps in synthesis of bile salts Used as detoxicants

55. Glu , Cys & Gly synthesise glutathione His changes to histamine on decarboxylation Serotonin is formed from Trp Gly is used in synthesis of heme Met acts as active methionine & helps in transmethylation Cys & Met are sources of sulphur Asp & Gln for pyramidine synthesis Asn & Glu for purine synthesis

56. References: Biochemistry by DM Vasudevan,Sreekumari S, 4 th edition. Tietz Fundamentals of Clinical Chemistry, 6th edition by Carl A. Burtis , Edward R. Ashwood , and David E. Bruns , editors. St Louis. Devlin's Textbook of Biochemistry,4 th Edition by Thomas M Devlin. Textbook of biochemistry by S atyanarayana , 4 th edition. Garrett & Grisham, Biochemistry, 4 th edition. Textbook Of Medical Biochemistry, Jaypee , Mn Chatterjee , Rana Shinde . Biochemistry by Pankaja Naik , Pankaja , Ph.D. Jaypee , 3 rd edition. World wide web.

57. To hold, as it were the mirror up to nature. - W illiam Shakespeare Thank You