AA Protein Enzyme

8/2/2019 AA Protein Enzyme

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Chapter 2:Biomolecules: Amino Acids and

Peptides

Genaro F. Alderite Jr.,MSERMMedical Biochemistry


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At physiological pHs (7.0 -7.4), boththe carboxyl and amino groups arecharged


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Phenylalaninehydroxylase


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arginase

urea


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Non-protein Amino Acids


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Examples of ClinicalAminoacidurias

Metabolic defects: Phenylketonuria (Phe),Tyrosinemias (Phe,Tyr), Maple Syrup UrineDisease (Leu, Val, Ile), Alcaptonuria (Tyr)

Absorption/transport defects: cystinuria (Cys),Hartnup disease , Fanconis Syndrome

These diseases are generally diagnosed fromindicators in the urine or plasma.


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BONDPEPTIDE


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Levels of Protein Structure


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General Properties of Amino Acids:

I. Physical Properties:1. White crystalline

2. Soluble in cold water, except cysteineand tyrosine.3. Most are insoluble in alcohol.4. Most are sweet like glycine, alanine,serine and proline.5. Others like leucine are tasteless;while some are bitter like arginine.


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II. Chemical Properties:1. Amino acids are amphoteric.2. They form esters with alcohol.3. Amino acids can be acetylated,benzylated or methylated.4. All amino acids except prolineand hydroxyproline react withnitrous acid with the liberation of nitrogen gas.


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Chapter 3:PEPTIDES AND PROTEINS

Genaro F. Alderite Jr.,MSERM

Medical Biochemistry


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Biomedical importance of proteins

in The Body:1. Enzymatic catalysis - almost all biological

reactions are enzyme catalyzed . Enzymes

are known to increase the rate of abiological reaction by a factor of 10 to the6th power! There are several thousand

enzymes which have been identified todate.


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2. Binding, transport and storage - small moleculesare often carried by proteins in the physiological

setting (for example, the protein hemoglobin isresponsible for the transport of oxygen totissues). Many drug molecules are partiallybound to serum albumins in the plasma.

3. Molecular switching - conformational

changes in response to pH or ligandbinding can be used to control cellularprocesses


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4. Coordinated motion - muscle is mostlyprotein, and muscle contraction is mediated by

the sliding motion of two protein filaments,actin and myosin.

5. Structural support - skin and bone arestrengthened by the protein collagen


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6. Immune protection - antibodies are proteinstructures that are responsible for reacting withspecific foreign substances in the body.

7.Generation and transmission of nerve impulses -some amino acids act as neurotransmitters ,which transmit electrical signals from one nervecell to another. In addition, receptors forneurotransmitters, drugs, etc. are protein in nature.

An example of this is the acetylcholine receptor ,which is a protein structure that is embedded inpostsynaptic neurons.


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8.Control of growth and differentiation --proteins can be critical to thecontrol of growth,cell differentiationand expression of DNA.

-For example, repressor proteins may bind to specific segments of

DNA, preventing expression and thusthe formation of the product of thatDNA segment.

-many hormones and growth factorsthat regulate cell function, such asinsulin or thyroid stimulatinghormone are proteins.


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Classification of Proteins:

1. Simple proteins- true proteins found abundantly inboth plants and animals.a. Albumins- are soluble in water and dilute

neutral solutions.- Members include serumalbumin, lactal albumin, andovalbumin.

b.Globulins- are soluble in neutral dilute salt solutionsbut not in water.- include legumin from peas,

myosinogen from muscles.


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c. Glutelins- soluble in dilute acids andalkalines but insoluble in neutral

solvents. - examples are glutenin fromwheat and oryzenin from rice.

d. Prolamines- are insoluble in ordinarysolvent but soluble in 70% alcohol at aboutneutral point.

- Present in plants such asgliadin from wheat, zein from corn, andhordein from barley.


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e. Histones- soluble in water, diluteacids and alkalines but not in diluteammonia.- not readily coagulated by heat- strongly basic and occur inthe tissues in the form of saltcombinations.- examples are globin fromhemoglobin, thymus histone andscobrone of mackerel.


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f. Protamines- contain smaller number of amino acids.

- strongly basic and formsoluble salts with strongmineral acids.- e.g salmin from salmonsperm

g. Scleroproteins- insluble in water andneutral solvents.

- e.g keratin of theepidermal tissues, elastin fromligaments and collagen fromhides, bones and cartilages


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2. Conjugated Proteins

a. Nucleoproteins- are combination of histones and protamines with nucleicacid.- soluble in dilute solutions of NaCl andcan be extracted from the tissues by theuse of this solvent.- typical examples are chromatin, andthe products obtain from glandulartissues and germ of grains.


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b. Glycoproteins- are compounds of proteinswith a carbohydrate component.

- they are utilized for lubricatingpurposes in view of their slimynature.

- mucin from saliva, tendomucoidfrom tendons and osseomucoidfrom bones belong to this group.

- are not digested in the GItract and used as protection.


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c. Phosphoproteins- have the prosthetic group(H 3PO 4) joined in the protein

molecule.- casein from milk and vitelline

of the egg yolk are rich in this type of

protein.

d. Chromoproteins- are protein compounds with

hematin or similar pigments in theirmolecule.- examples are hemoglobin, cytochromes

and rhodopsin.


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e. Lipoproteins- have fatty substancescombined with their moleculeslike lecithin, cephalin, etc.- they are present in the blood

serum, brain tissues, cell nuclei,egg yolk and milk.


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3. Derived Proteins- substances formed fromsimple and conjugated proteins.

a. Primary protein derivatives- are proteinswhich have undergone intramolecular

rearrangement through the hydrolyticaction of certain physical and chemicalagents.- They are synonymous withdenatured proteins.

1 P t i l bl b t


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a1. Proteans- are insoluble substancesresulting from the preliminary action of water, dilute acids, or enzymes.- myosan from myosin and edestanfrom edestin are good examples.

a2. Metaproteans- are product of furtherhydrolysis.- soluble in weak acids andalkalies, but insoluble in neutralsalt solutions.

-acid metaproteans(acid albuminate);alkali metaproteans (alkali albuminate


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a3. Coagulated proteins- are insolubleproducts resulting from either theaction of heat, alcohol, ultravioletare or even simple mechanicalshaking.-cooked egg albumin, cooked

meat,etc.


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b. Secondary protein derivatives- are products of more extensive hydrolysis.

b1. Primary proteoses- are soluble in water,precipitated by concentrated nitric acid.

- not coagulated by heat.b2. Secondary proteoses- precipitated only bycomplete saturation with ammonium sulfate

but not with nitric or picric acid.


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b3. Peptones- are insoluble in water, notcoagulated by hear and not precipitated by

ammonium sulfate but by certain alkaloidalreagents such as phosphotungstic and tannicacids.

b4. Peptides- are combinations of two or moreamino acids , the carboxyl group of one beingunited with the amino group of the other.

-same properties with peptones.-ex: di,tri,tetra, penta,poly


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Protein Structure Levels

PRIMARY: the linear sequence ofamino acids linked together by peptide

bonds SECONDARY: regions within

polypeptide chains with regular,

recurring, localized structure stabilizedby H-bonding between constituentamino acid residues


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Secondary Protein Structure: a-helix

& b-sheet


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Super-secondary structure examples


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TERTIARY: the overall three-dimensionalconformation of a protein

QUATERNARY: the three-dimensionalconformation of a protein composed of

multiple polypeptide subunits

THE PRIMARY AMINO ACID SEQUENCEIS THE ULTIMATE DETERMINANT OFFINAL PROTEIN STRUCTURE


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Myoglobin b -subunit Hemoglobin


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Structure of Myoglobin and

Hemoglobin The amino acid sequences of myoglobin and

hemoglobin are similar (or, highly conserved) but

not identical Their polypeptide chains fold in a similar manner Myoglobin is found in muscles as a monomeric

protein; hemoglobins are found in matureerythrocytes as multi-subunit tetrameric proteins.Both are localized to the cytosol


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Stabilizing Forces

1. Electrostatic/ionic2. Hydrogen bonds3. Hydrophobic interactions4. Disulfide bonds


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1. Electrostatic/ionic 3. Hydrophobic interactions2. Hydrogen bonds 4. Disulfide bonds


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Biochemical Methods to Analyze

Proteins Electrophoresis Chromatography: Gel filtration, ion

exchange, affinity Mass Spectrometry, X-ray

Crystallography, NMR


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Gel filtration

Separation is based on protein size.Dextranor polyacrylamide beads of uniform

diameter are manufactured with differentpore sizes.

Depending on the sizes of the proteins to be

separated, they will enter the pore if smallenough, or be excluded if they are too large.


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Ion Exchange Chromatography

Separation of proteins based on the netcharge of their constituent amino acids.

Different salt concentrations can be used toelute the bound proteins into tubes in a

fraction collector. Resins for binding (+) or(-) charged proteins can be used


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Affinity Chromatography

Based on the target proteins ability to bind aspecific ligand, only proteins that bind to

this ligand will be retained on the columnbead.

This is especially useful for immunoaffinity

purification of proteins using specificantibodies for them.


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Physical and Chemical Properties of


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Physical and Chemical Properties of Proteins:

1. When pure, proteins are generally tastelessexcept with hydrolates.

2. Mostly colorless.3. Insoluble in fat solvents and present varied

degrees of solubility in water, salt solution, dilute

acids and alkalies.4. Proteins are amphoteric.5. Proteins are very reactive and highly specific.


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Solubility of Proteins: (Major

influences)

1. The effect of neutral salt.

2. The effect of pH.3. The effect of organic solvents.


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Actions of Heat

- when burned, proteins decompose and liberatea characteristic odor of burned hair or feather.

- solutions of proteins when heated between 38-60 degrees centigrade, undergo slight

intramolecular rearrangements.( DENATURATION)


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Precipitations:

1. By acids2. By salts of Heavy metals

3. By alcohol

Hydrolysis:

1. Dilute acids; alkalies or enzymes- due to addition of elements in the peptide bond/ linkage.


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CLINICAL SIGNIFICANCE OF

PROTEIN:1. The substitution of a hydrophobic amino

acid (V) for an acidic amino acid (E) in the

-chain of hemoglobin results in sickle cellanemia (HbS). This change of a singleamino acid alters the structure of hemoglobin molecules in such a way that

the deoxygenated proteins polymerize andprecipitate within the erythrocyte, leading totheir characteristic sickle shape.


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b. Osteogenesis imperfecta also encompasses


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g p pmore than one disorder. At least four biochemicallyand clinically distinguishable maladies have beenidentified as osteogenesis imperfecta, all of whichare characterized by multiple fractures and resultantbone deformities.

c. Marfan syndrome manifests itself as adisorder of the connective tissue and was originallybelieved to be the result of abnormal collagens.However, recent evidence has shown that Marfansyndrome results from mutations in the extracellularprotein, fibrillin, which is an integral constituent of the non-collagenous microfibrils of the extracellularmatrix.


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3. Several forms of familial hypercholesterolemiaare the result of genetic defects in the gene

encoding the receptor for low-densitylipoprotein (LDL). These defects result in thesynthesis of abnormal LDL receptors that are

incapable of binding to LDLs, or that bindLDLs but the receptor/LDL complexes are notproperly internalized and degraded. The

outcome is an elevation in serum cholesterollevels and increased propensity toward thedevelopment of atherosclerosis.

4 A b f t i t ib t t ll l


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4. A number of proteins can contribute to cellulartransformation and carcinogenesis when their

basic structure is disrupted by mutations in theirgenes. These genes are termed proto-oncogenes.For some of these proteins, all that is required toconvert them to the oncogenic form is a singleamino acid substitution. The cellular gene, RAS, isobserved to sustain single amino acid substitutionsat positions 12 or 61 with high frequency in colon

carcinomas. Mutations in RAS are most frequentlyobserved genetic alterations in colon cancer.


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MARAMINGSALAMAT!

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AA Protein Enzyme