Amino Acid, Peptide, Protein

Harliansyah, Ph.D Head Dept of Biochemistry, FKUY 2010

Email : ianshr2001@yahoo.com

A.

Physical Properties
1. Solubility - soluble in water and insoluble in
organic solvents

2. Melting Points - melt at higher temperatures >

often 200C

3. Taste

sweet (Gly, Ala, Val) tasteless (Leu) bitter (Arg, Ile) Sodium Glutamate

salt of Glutamic Acid flavoring agent

4. Optical Properties
- Assymetric a carbon atom is attached to 4 different groups

exhibiting optical isomerism

All AA except Glycine possess optical isomers due to asymmetric -carbon atom Some AA (Isoleucine, Threonine) 2nd asymmetric carbon

D- and L- forms of AA based on the structure of glyceraldehyde

5. Amino acids as ampholytes

can donate a proton or accept a proton AA contain both acidic (-COOH) and basic (-NH2) groups

Zwitterion or dipolar ion:

Zwitter hybrid from German word means

Zwitter ion (or dipolar ion) a hybrid molecule containing (+) groups

and (-) ionic

AA rarely exist in a neutral form with free carboxylic (-COOH) and free Amino (-NH2) groups Strongly acidic pH (low pH) AA (+) charged (cation)
Strongly alkaline pH (high pH) AA (-) charged (anion) Each AA has a characteristic pH (e.g. Leucine, pH 6.0), at which it carries both (+) and (-) charges and exist as zwitterion

Chemical Properties General Reactions mostly due to the 2 functional groups Reactions due to - COOH group 1. AA from salts (-COONa) with bases and esters (-COOR) with alcohols 2. Decarboxylation - AA undergo decarboxylation to produce 3. Reaction with Ammonia - the carboxyl group of dicarboxylic AA reacts Asparatic Acid + NH3 Asparagine Glutamic Acid + NH3 Glutamine with NH3 to form amide

corresponding amines

Reactions due to -NH2 group 4. The Amino groups behave as bases and combine with acids (e.g. HCl) to form salts (-NH3 + Cl) 5. Reaction with NINHYDRIN (Ruhemanns blue purple)

6. Colour reactions of Amino Acids - AA can be identified by specific colour reactions Color Reactions of proteins / AA Specific group or AA Two peptide linkages -Amino acids Indole ring of aromatic AA (Trp) Phenolic Group (Tyr) Aromatic ring (Phen, Tyr,Trp)

Reaction
1.

2.
3. 4. 5.

Biuret Reaction Ninhydrin Reaction Hopkins Cole Reaction Millions reaction Xanthoprotein Reaction

Millons Test

6. Sakaguchi Reaction 7. Nitroprusside Reaction 8. Paulys test 9. Sulfur test 10. Folin Coicalteaus
Fohls Reaction

Guanidino Group (Arg) Sulfhydryl groups (Cys) Imidazole ring (His) Sulfhydryl groups (Cys) Phenolic groups test (Tyr)

7. Transamination - important reaction in AA metabolism - transfer of an amino group from an amino acid to a keto acid to form a new AA 8. Oxidative deamination - AA undergo oxidative deamination to liberate free ammonia

Characteristics of Peptide Bonds

1.

2.
3. 4. 5.

6.

Rigid Planar Partial double bond in character Generally exists in transconfiguration Both _C = O and _NH2 groups of peptide bonds are polar Involved in hydrogen bond formation

Writing of Peptide Structures

The peptide chains are written with the free Amino end (N terminal residue) at the left, and the free carboxyl end (C terminal residue) at the right. The AA sequence is read from N terminal end to C terminal end Incidentally the protein biosynthesis also starts from the N terminal Acid.

Naming of Peptides

For naming peptides, the AA suffixes ine (glycine), - an (tryptophan) ate (glutamate) are changed to yl with the exception of C terminal AA.

A tripeptide composed of an N terminal glutamate, a cysteine and a C terminal glycine is called: glutamyl cysteinyl - glycine

Polypeptide Chains
- The linking together of many AA by peptide bonds produces polypeptide chains.

a.Residues - AA, when in polypeptide chains, are customarily referred to as residues. b.Large peptide chains - protein polypeptide chains are typically more than 100 AA residues long. The backbone of the chain is a recurring

Bovine Insulin: the first sequenced protein

Human: Thr-Ser-Ile Cow: Ala-Ser-Val Pig: Thr-Ser-Ile Chiken: His-Asn-Thr

The role of side chain in the shape of proteins

Where is water?

Hydrophilic

Hydrophobic

Amino acid substitution in proteins from different species

Conservative

Substitution of an amino acid by another amino acid of similar polarity

(Val for Ile in position 10 of insulin)

Non conservative

Substitution involving replacement of an amino acid by another of different polarity

(sickle cell anemia, 6th position of hemoglobin replace from a glutamic acid to a valine induce precipitation of hemoglobin in red blood cells)

Invariant residues

Amino acid found at the same position in different species

(critical for for the sructure or function of the protein)

Biuret Reaction

Stabilized by hydrogen bonds H- bonds are between CO and NH groups of peptide backbone H-bonds are either intra- or intermolecular 3 types : a-helix, b-sheet and triple-helix

Two type of b Sheet structures

An anti paralellel b sheet

A paralellel b sheet

Triple helix of Collagen

Peptides of Physiologic Importance

1.

Glutamine (Glutathione) - a tripeptide composed of 3 AA - gamma glutamyl cysteinyl glycine - wildly distributed in nature - exists in reduced or oxidized states

Functions:
a) As a coenzyme for certain enzymes as prostaglandin PGE2 synthase glycoxylase b) Prevents the oxidation of sulfhydryl groups of several proteins to disulfide groups c) In association with glutathione reductase participates in the formation of correct disulfide bonds in several protiens d) In erythrocytes - maintains RBC membrane structure and integrity - protects hemoglobin from getting oxidized by agents such as H2O2

e) Involved in the transport of AA in the intestine and kidney tubules via delta glutamyl cycle or Meister cycle
f) Involved in the detoxification process g) Toxic amounts of peroxidases and free radicals produced in the cells are scavanged by glutathione peroxidase ( a selenium containing enzyme).

2. Thyrotropin Releasing Hormone (TRH) - a tripeptide secreted by hypothalamus Function: Stimulate pituitary gland to release thyrotropic hormone 3. Oxytocin - contains 9 AA (nonapeptide) - hormone secreted by posterior pituitary gland Function: Stimulate contraction of the uterus muscle during delivery

Stimulate contraction of muscle in breasts for milk ejection

Oxytocin and vasopressin are two peptide hormones with very similar structure, but with very different biological activities. Interestingly, their structures only differ by one amino acid residue (the hydrophobic LEU number 8 in oxytocin is replaced by a hydrophilic ARG residue in vasopressin). Oxytocin is a potent stimulator of uterine smooth muscle, and also stimulates lactation. Vasopressin, also know as antidiuretic hormone (ADH), has no effect on uterine smooth muscle, but causes reabsorbtion of water by the kidney, thus increasing blood pressure.

4. Vasopressin (ADH antidiuretic hormone) - also a nonapeptide - produced by posterior pituitary gland Function: Stimulates kidneys to retain water and thus increases the blood pressure 5. Angiotensins - Angiotensin 1 a decapeptide (10AA) which is converted to angiotensin II (8AA) Function: For the release of aldosterone from adrenal gland

6. Methionine Enkephalin - a pentapeptide found in the brain and has opiate like function. Function: It inhibits the sense of a pain.
7. Bradykinin and Kallidin - nona and decapeptides respectively - produced from plasma proteins by snake venom enzymes Function: Powerful vasodilators

8. Peptide Antibiotics - Antibiotics such as Gramicidin, Bacitracin, tyrocidin and Actinomysin peptide in nature
9. Dipeptide aspartame - Consists of aspartate and phenylalanine - acts as Sweetener ~ used by diabetic patients 10. Gastrointestinal Hormones - Gastrin, Secretin & etc. - gastrointestinal peptides serving as hormones

- they differ in their physicochemical properties which ultimately determine the characteristics of proteins

The Proteins speak:

We are the basis of structure and function of life; Composed of twenty amino acids, the building blocks; Organized into primary, secondary, tertiary and quaternary structure; Classified as simple, conjugated and derived proteins.

- 4 different forces stabilize the tertiary structure of globular protein i. Hydrogen bonding between R groups of residues in adjacent loops of the chain ii. Ionic attraction between oppositely charged R groups iii. Hydrophobic interactions iv. Covalent cross-linkages (via intrachain cystein residues)

Function of proteins
Enzymatic catalysis Transport and storage (the protein hemoglobin, albumins) Coordinated motion (actin and myosin). Mechanical support (collagen). Immune protection (antibodies) Generation and transmission of nerve impulses - some amino acids act as neurotransmitters, receptors for neurotransmitters, drugs, etc. are protein in nature. (the acetylcholine receptor), Control of growth and differentiation transcription factors Hormones growth factors ( insulin or thyroid stimulating hormone)

Proteins are the most important buffers in the body.

Why?
(a) Protein molecules possess basic and acidic groups which act as H+ acceptors or donors respectively if H+ is added or removed.

(a)

Proteins are the most important buffers in the body. They are mainly intracellular and include haemoglobin.

(b) The plasma proteins are buffers but the absolute amount is small compared to intracellular protein. (c) Protein molecules possess basic and acidic groups which act as H+ acceptors or donors respectively if H+ is added or removed.

Many proteins (thousands!) present in blood plasma Proteins contain weakly acidic (glutamate, aspartate) and basic (lysine, arginine, histidine) side chains (or R groups) At neutral pH, only histidine residues (containing imidazole R group with pKa ~ 6.0) in proteins can act as a buffer component Haemoglobin with 38 histidine/tetramer is a good buffer N-terminal groups of proteins (pK a ~ 8.0) can also act as a buffer component

Classes of protein
1 - Structural protein : Keratin, Collagen (give support to cells) 2 - Dynamic protein : Hormone, enzyme (for catalytic purpose) - Based on the structure, protein can be divided to : * Fibrin : Blood clotting

* Fibrous : Myosin (from muscle)

* Globular : Half sphere form/structure eg. Enzyme - Size : Varied - depending on functions - 1 amino acid = 110 Daltons - Most protein are highly folded

Fibrous and Globular Proteins

- There are also protein which its polypeptide chains are tightly folded into a spherical or globular shape

Example of globular protein : Lysozyme molecule with its tightly bound polysaccharide substrate (color)

Enzyme Catalysis

Transport and storage - small molecules are often carried by proteins in the physiological setting
(for example, the protein hemoglobin is responsible for the transport of oxygen to tissues). Many drug molecules are partially bound to serum albumins in the plasma.

The binding of oxygen is affected by molecules such as carbon monoxide (CO) (for example from tobacco smoking, cars and furnaces). CO competes with oxygen at the heme binding site. Hemoglobin binding affinity for CO is 200 times greater than its affinity for oxygen, meaning that small amounts of CO dramatically reduces hemoglobin's ability to transport oxygen. When hemoglobin combines with CO, it forms a very bright red compound called carboxyhemoglobin. When inspired air contains CO levels as low as 0.02%, headache and nausea occur; if the CO concentration is increased to 0.1%, unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked by CO. 3-dimensional structure of hemoglobin. The four subunits are shown in red and yellow, and the heme groups in green.

Coordinated motion - muscle is mostly protein, and muscle contraction is mediated by the sliding
motion of two protein filaments, actin and myosin.

Platelet activation is a controlled sequence of actin filament: Severing Uncapping Elongating Cross linking That creates a dramatic shape change in the platelet

Platelet before activation

Activated platelet

Activated platelet at a later stage than C)

Mechanical support - skin and bone are strengthened by the protein collagen.

Abnormal collagen synthesis or structure causes dysfunction of

cardiovascular organs, bone, skin, joints eyes
Refer to Devlin Clinical correlation 3.4 p121

Immune protection - antibodies are protein structures that are responsible for reacting with specific
foreign substances in the body.

Generation and transmission of nerve impulses Some amino acids act as neurotransmitters, which transmit electrical signals from one nerve cell to another. In addition, receptors for neurotransmitters, drugs, etc. are protein in nature. An example of this is the acetylcholine receptor, which is a protein structure that is embedded in postsynaptic neurons.

GABA: gamma Amino butyric acid Synthesised from glutamate

GABA acts at inhibitory synapses in the brain. GABA acts by binding to specific receptors in the plasma membrane of both pre- and postsynaptic neurons. Neurotransmetter

Membrane transport proteins

Protein degradation:

Disease and protein folding:

Disease
Example: Neurodegenerative diseases

Digestion of Proteins
1. Mouth - food is chewed 2. Stomach: Gastrin - triggers chief cells to release HCl and pepsinogen 3. Small intestines: Secretin-stim. release of pancreatic juices Activation of proteolytic enzymes: Trypsinogen trypsin Chymotrypsinogen chymotrypsin Proelastase elastase Endo & exopeptidases activated

Products of enzyme action = amino acids Dipeptides & small peptides AA Greater amount of AA in portal blood is in the form of alanine STOMACH

Protein digestion starts in the stomach dietary proteins become denatured by gastric acid important for protein digestion because proteins are poor substrates for proteases

ACID ENVIRONMENT is required for action of pepsin

PEPSIN

A protease that works optimally at pH 2 a carboxyl protease acts mostly as endopeptidase does not cleave at random prefers peptide bonds formed by amino group of aromatic AA major breakdown products are not free AA but a mixture of oligopeptides known as peptones

INTESTINE As acidic stomach contents reach duodenum rapidly neutralized by HCO3 in pancreatic secretions. Proteolytic enzymes from pancreas include: 1. Trypsin a serine protease endopeptidase specific for CO side of basic AA

2. Chymotrypsin a serine protease endopeptidase specific for CO side of hydrophobic AA 1 & 2 = degrade peptones to smaller peptides 3. Carboxypeptidase A: hydrophobic AA at C terminal 4. Carboxypeptidase B basic AA at C terminus