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AMINO ACIDS AND PEPTIDESMaureen Baroro- De Guzman, MDBIOMEDICAL IMPORTANCEProvide the monomer units of polypeptide chains of proteinsParticipate in cellular functions ( nerve transmission, porphyrins, purines, pyrimidines, urea)Peptides: short polymers of proteins (role in neuroendocrine system)D- and L- amino acids: by microorganisms (therapeutic value)Humans: lack the capability to synthesize 10 of the 20 common L-amino acids, diet must contain adequate quantities of these essential amino acidsPROPERTIES OF AMINO ACIDSTABLE 3-1 PP 15

AMINO ACIDS: BUILDING BLOCKS FOR PROTEINSOrganic compound that contains both an amino (-NH2) group and a carboxyl (-COOH) group

amino acids :amino and carboxyl groups are attached to the carbon; AA in proteins

The Genetic Code Specifies 20 L- Amino AcidsRedundancy of 3- letter genetic code: limits the available codons to the 20 L-a amino acids

Some proteins: contain additional amino acids that arise by modification of an amino acid already present in a peptide Conversion of peptidyl proline and lysine to 4-hydroxyproline and 5- hydroxylysinePeptidyl glutamate to - carboxyglutamate

Modifications: extend the biologic diversity of proteins by altering their solubility, stability and interaction with other proteins

Selenocysteine, the 21st L- amino acid? Selenocysteine: L- a amino acid found in peroxidases and reductasesParticipates in the catalysis of electron transfer reactionSelenium atom replaces the sulfur of its structural analog cysteineInserted into polypeptides during translation: commonly referred to as the 21st amino acidUnlike the other 20 amino acid: not specified by the 3-letter codon

THE ESSENTIAL AMINO ACIDSamino acids needed by the body that must be obtained from dietary sources because it cannot be synthesized within the body from other substances in adequate amountsArginine: required for growth in children but not required in adults

Only L- Amino Acids Occur in Proteins amino acids are chiral (except glycine)Share absolute configuration of L-glyceraldehyde : L- amino acids Free amino acids: role in metabolic processes ornithine, citrulline, argininosuccinate in urea synthesisTyrosine: thyroid hormone synthesisGlutamate: neurotransmitter biosynthesisD- Amino acids that occur naturally: D- serine and aspartate in brain tissueD- alanine and glutamate: cell walls of G+ bacteriaIn certain peptides and antibiotics produced by bacteria , fungi, reptiles and nonmammalian species

Acid- Base Properties Of Amino AcidsPure form: white crystalline solids with high decomposition pointsMost are not very water soluble due to strong intermolecular forces within in their crystal structuresAA are charged species both in the solid state and in solutionBoth an acidic group (-COOH) and a basic group (-NH2) are present

Neutral Solution: carboxyl groups have a tendency to lose protons (H+) producing a negatively charged species-COOH -COO- + H+

Neutral solution: amino groups have a tendency to accept protons (H+) producing a positively charged species-NH2 + H+ - +NH3

-COOH groups donates a proton to the NH2 of the same amino acid; internal base reaction with a net result

called a Zwitterion (German meaning: double ion)A molecule that has a positive charge on one atom and a negative charge on another atom but which has no net chargeNet charge: zeroStrong intermolecular forces between the positive and the negative centers: high melting points of amino acids

Zwitterion ion structure change: pH of solution containing an amino acid is changed from neutral either to acidic (low pH) by adding an acid such as HClbasic (high pH) by adding a base such as NAOH

Acidic Solution: zwitterion accepts a proton (H+) to form a positively charged ion

Basic Solution: the NH3 of the zwitterion loses a proton and a negatively charged species is formed

In solution, 3 different solutions can exist (zwitterion, negative and positive ion ion)3 species in equilibrium with each otherEquilibrium shifts with pH changeOverall equilibrium process

Ability of amino acids to react with both H30+ and OH- ions: can function as buffers

Guidelines for amino acid form as a function of solution of pH Low pH: All acid groups are protonated (-COOH). All amino groups are protonated (- +NH3)

High pH: All acid groups are deprotonated (-COO-). All amino groups are deprotonated (-NH2).

Neutral pH: All acid groups are deprotonated (-COO-). All amino groups are protonated (- +NH3).

Assumption that that the side chain (R chain) of an amino remains unchanged in solution as the pH is varied (for neutral amino acids)

Acidic and Basic Amino acids: side chain can also acquire a charge because it contains an amino or a carboxyl group that can gain of lose a proton respectively

Protonated: gain of H+ ionDeprotonated : loss of a H+ ion

With an extra site that can be protonated or deprotonated, acidic and basic amino acids have four charged forms in solution

Four forms of aspartic acid

Side chain carboxyl groups are weaker acids that a- carbon carboxyl groups

pKa values express the strengths of Weak AcidspKa: acid strengths of weak acidsMolecules with multiple dissociable protons: pKa for each acidic group is designated by replacing a subscript a with a numberNet charge on amino acid: algebraic sum of all the positively and negatively charged groups present- depends upon the pKa values of its functional groups and on the pH of the surrounding mediumAltering the charge by varying the pH facilitates the physical separation of amino acids, peptides and proteins

At its isoelectric pH, an Amino acid bears no net chargeZwiterrions: isoelectric species, equal number of negative charges (electrically neutral)Isoelectric pH (pI): pH midway between pKa values on either side of the isoelectric speciesExample: Alanine with 2 dissociating groups1st pKa (R-COOH): 2.352nd pKa (R-NH3+): 9.69pI: pK1 +pK2 = 2.35+ 9.69 =6.02 2 2

Polyfunctional acids: pI is also the pH midway between the pKa values on either side of the isoionic speciesAlso apply to Polyprotic acids (proteins): regardless of the number of dissociating groups presentLaboratory: pI guides selection of conditions for electrophoretic separationsEx: Elecgtrophoresis at pH 7.0: separate 2 molecules with pI values 6.0 and 8.0Because at pH 7, the molecule with a pI 6.0 with have a net positive charge, pI 8.0 with a negative charge

pKa values vary with the environmentpKa values of the R groups of free amino acids: provide only an approximate guide to the pKa values of the same amino acids in proteinsPolar environment: favors charged form (R-COO or R-NH3+), Non polar environment: favors uncharged form (R-COOH and R-NH2); raises the pKa of a carboxyl group (making it a weaker acid) but lowers that of an amino group (making it a weaker acid)Presence of adjacent charged groups: reinforce or counteract solvent effectpKa of a functional group: will depend on its location with in a given proteinVariations in pKa can encompass whole pH units (table 3-2)

Solubility of Amino Acids reflects their Ionic CharacterCharged functional groups of amino acids: ensure that they are readily solvated by- and thus soluble in- polar solvents such water and ethanolBut insoluble in nonpolar solvents (benzene, hexane)Amino acids: do not absorb visible light, colorlesTyrosine, Phe, Tryp: absorb wavelength (250-290 nm) UVTyrp: Absorbs UV light 10x more efficiently than phe or Tyr, makes major contribution to the ability of most proteins to absorb light in the region of 280 nm

THE - R GROUPS DETERMINE THE PROPERTIES OF AMINO ACIDSGlycine: smallest amino acid, accomodated in places inaccessible to other amino acids, occur where peptides bend sharplyHydrophobic R groups (Ala, Val, Leu, Iso and aromatic R groups of Phe, Tyr, Tryp): occur primarily in the interior of cytosolic proteinsCharged R groups of basic and acidic amino acids: stabilize specific protein conformations via ionic interactions or salt bridgesSuch interactions functions in charge relay systems during enzymatic catalysis and electron transport in respiring mitochondria

Histidine: unique role, pKA of its imidazole proton permits it to function at neutral pH as either a base of an acid catalyst

Primary alcohol group of Ser and thioalcohol of Cysteine: excellent nucleophiles, function during enzymatic catalysis

Secondary alcohol group of Threonine: good nucleophile, doe not fulfill the same role of Ser and Cysteine

-OH groups of ser, try and threonine: regulation of activity of enzymes whose catalytic activity depends on the phosphorylation state of these residuesFUNCTIONAL GROUPS DICTATE THE CHEMICAL REACTIONS OF AMINO ACIDSEach functional group of an amino acids: exhibits all of its characteristic chemical reactionCarboxylic acid groups: formation of esters, amides and acid anhydridesAmino groups: acylation, amidation and esterification-Oh and Sh groups: oxidation and esterificationFORMATION OF PEPTIDE BOND: most important reaction of amino acids

Amino Acid Sequence Determines Primary Structureprimary structure: Number and order of all of the amino acid residues in a polypeptide chainAminoacyl residues: amino acids present in peptidesNamed by replacing the ate or ine suffixes of free amino acids with yl (alanyl, aspartyl, lysyl)Peptides: named as derivatives of the carboxyl terminal aminoacyl residuesExample: Lys-Leu-Tyr-GlnLysyl-Leucyl-Tyrosyl-Glutamine-ine ending of Glutamine indicate that its a-carboxyl group is not involved in the peptide bond formation

PEPTIDE unbranched chain of amino acids, each joined to the next by a peptide bond

Classified by the number of peptide bondsDi- , tri-, oligo

Polypeptide: long, unbranched chain of amino acids, each joined to the next by a peptide bond

NATURE OF PEPTIDE BOND Carboxyl group of one amino acid interacts with the amino group of the other amino acid

Products: water and molecule containing 2 amino acids linked by an amide bondDirectionality: N- terminal end C- terminal end

NATURE OF PEPTIDE BOND Sequence of amino acids in a peptide is written with the N- terminal amino acid on the left

Amino acid residue: portion of an amino acid structure that remains after release of H2O, when an amino acid participates in peptide bond formation as it becomes part of the peptide chain

Structural Formula: may be written in full or by the standard 3-letter AA abbreviations; AA at the N-terminal end of the peptide is always written on the leftE.g. Glyc- Ala- Ser

PEPTIDE NOMENCLATUREIUPAC Rules for Naming small peptidesThe C- terminal amino acid residue keep its full amino acid nameAll of the other amino acid residues have names that end in yl. The yl suffix replaces the ine or ic acid ending of the amino acid name, except for Tryptophan (tryptophyl), cysteine (cysteinyl), glutamine (gluatminyl) and asparagine (asparaginyl)The amino acid naming sequence begins at the N-terminal amino acid residue

Assign IUPAC names:

Glu- Ser- AlaGly- Tyr- Leu- Val

Answers:

GlutamylserylalanineGlycyltyrosylleucylvaline

Some peptides contain unusual amino acidsMammals: peptide hormones typically contain only a- amino acids of proteins linked by standard peptide bondsOthers: contain nonproteins amino acids, derivatives of amino acids or amino acids linked by an atypical peptide bondEx: amino terminal of glutathione (participates in folding and in the metabolism of xenobiotics): linked to cysteine by a non- a peptide bond

Peptides are polyelectrolytesPeptide bond: uncharged at any pH of physiologic pHFormation of peptides from amino acids: accompanied by a net loss of one positive and one negative charge per peptide bond formedCharged at physiologic pH: carboxyl and amino terminal groups and where present, their acidic or basic R groupsAmino acids: the net charge on a peptide depends on the pH of its environment and the pKa values of its dissociating groups

y32The Peptide Bond Has Partial Double- Bond CharacterSingle bond linked to the a-carboxyl and a-nitrogen atoms: exhibits partial double bondThus, no freedom of rotation about the bond that connects the carbonyl and the nitrogen of a peptide bondO, C, N, H are co planarImposed semi-rigidity of the peptide bond: important consequences for the manner in which peptides and proteins fold to generate higher orders of structure

Noncovalent Forces Constrain Peptide ConformationsFolding of a peptide bond: coincident with its biosynthesisPhysiologically active conformation of : reflects the collective contributions of the amino acid sequences, steric hindrance, non covalent interactionsCommon conformations: a- helices and B- pleated sheets

Analysis of the Amino Acid Content of Biologic MaterialsDetermine the identity and quality of each amino acid in a sample of biologic material: necessary to hydrolyze the peptide bonds that link the amino acids together by treatment with hot HClResulting mixture of free amino acids: treated with 6-amino-N-hydroxysuccinimidyl carbamate which reacts with their a-amino groups to form fluorescent dreivatives that are separated and identified using high pressure liquid chromatographyNinhydrin : used for detecting amino acids and a yellow adduct with the imine groups of proline and hydroxylproline