Amino Acids, Proteins and Enzymes

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Amino Acids, Proteins and Enzymes

• Amino acids are the building blocks of proteins,
  20 amino acids are found in proteins
• They are called a amino acids attached to the
  alpha carbon is a H, NH2, COOH and R, four
  different groups
• The a carbon is a chiral carbon in all the amino
  acids except glycine (Gly)
• Amino acids that are not synthesized in the body
  and must be obtained from the diet are called
  essential amino acids (10)
• All naturally occuring amino acids in the human
  body are the L- enantiomers
• The peptide bonds that link amino acids in a
  protein are amide bonds.

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Amino acids in proteins have L-configuration
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Amino Acids

• In body fluids the amino acids exist in the
  dipolar form called a zwitterion which has a net
  charge of zero.
• The isoelectric point, pI, is the pH at which the
  amino acid has a net charge of zero.
• R-CH-COOH ? R-CH-COO-
• NH2 NH3

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Amino Acids as Acids Bases

• In a solution more acidic than the pI, lower pH,
  COO- acts as a base and accepts a H which gives
  an overall positive charge to the amino acid.
• In a solution more basic than the pI, higher pH,
  NH3 acts as an acid and loses an H which gives
  an overall negative charge to the amino acid.

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Zwitterions

• pH lt pI pH pI pH gt pI
• At a pH lt the pI they have a positive charge.
• At a pH gt the pI they have a negative charge.
• Acidic amino acids have low pI values as the
  extra -COO-
• Group must be neutralized first.
• Basic amino acids have high pI value as the extra
  NH3
• Group must be neutralized first.

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Classification of Amino Acidsby the polarity of
the side chain

• Hydrophobic (water fearing)
• Nonpolar side chains
• Gly, Ala, Val, Leu, Ile, Phe, Met, Pro, Trp
• Hydrophilic (water loving)
• Polar
• Neutral Ser, Thr, Tyr, Cys, Gln, Asn
• Acidic Glu, Asp
• Basic Arg, Lys, His

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Formation of a Peptidepeptide bonds amide bonds

• Peptide bonds join amino acids to form peptides
  by forming an amide bond between the carboxyl
  group of one amino acid and the amino group of
  the second amino acid.
• In a peptide, the free amino acid written on the
  left is called the N terminal and the free
  carboxyl group of the last amino acid is called
  the C terminal.
• In a peptide each amino acid is named with a yl
  ending followed by the full name of the amino
  acid at the C terminal.
• Histidylglycylglutamylalanine (HisGlyGluAla)

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Small Peptides

• With three AAs can get six possibilities, e.g. if
  add serine
• Glycylalanylserine (GAS) Serylalanylglycine
  (SAG)
• also AGS SGA GSA or ASG. With four AAs get 24
  possibilities, e.g. add proline
• PGAS GPAS GAPS GASP
• PSAG SPAG SAPG SAGP
• PAGS APGS AGPS AGSP
• PSGA SPGA SGPA SGAP
• PGSA GPSA GSPA GSAP
• PASG APSG ASPG ASGP
• The large number of possibilities is why proteins
  can have so many different sequences.
• The hormone angiotensin has the sequence
  DRVYIHPF these eight amino acids can be arranged
  in 40,320 ways.

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Proteins

• Proteins are long chains of more than 50 amino
  acids
• Proteins perform many functions in the body
• Structural
• Contractile
• Transport
• Storage
• Hormone
• Enzyme
• protection

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Levels of Protein Structure

• Primary Structure
• Provides the amino acid sequence held together by
  peptide bonds
• Human insulin has two polypeptide chains held
  together by disulfide bonds formed by the side
  chains of the cysteine amino acids in each of the
  chains

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Levels of Protein Structure

• Secondary Structure
• Alpha helix (myosin, actin, colicin Ia)
• a-helix - a right-handed coil of AAs with 3.6 AAs
  per turn, the side chains are on the outside
• Beta pleated sheet (silk) folded, parallel chains
• Triple helix (tropocollagen) three strands woven
  
• together like a braid. Left-handed helix.
• Is a result of hydrogen bonding between amino
  acids within the protein.

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Levels of Protein Structure

• Tertiary Structure
• Protein folds into a compact shape stabilized by
  interactions between the R groups.
• Interactions that stabilize the tertiary
  structure
• Hydrophobic attractions between nonpolar groups
  in the interior of the molecule
• Hydrophilic attractions between polar or
  ionized groups and water on the exterior of the
  molecule
• Salt bridges ionic attractions between ionized
  acidic and basic amino acids
• Hydrogen bonds between H and O or N between the
  same and/or different chains
• Disulfide bonds strong covalent bonds between
  sulfur atoms of two cysteine amino acids between
  the same and/or different chains

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Levels of Protein Structure

• Tertiary Structure
• Globular protein soluble in water, compact,
  roughly spherical, used by cell proteins, 3-D
  structure is tertiary.
• Albumins, globulins
• Myoglobin is a storage protein, stores oxygen in
  
• skeletal muscle. It has one
  heme.
• Fibrous protein long, thin, fiber-like shapes
  (filamentous), insoluble in water, used by cells
  and connective tissues
• a ß keratins hair, wool, skin, nails, silk,
  collagen, myosin.

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Levels of Protein Structure

• Quaternary Structure
• A combination of two or more protein subunits to
  form a larger, biologically active protein. The
  subunits are held together by the same
  interactions found in tertiary structure.
• Hemoglobin
• Protein with a globular structure, it is an
  oxygen transport protein
• Has 4 protein chains two alpha and two beta
• 4 hemes
• In a sickle-cell anemia the hemoglobin molecules
  clump together into insoluble fibers

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Protein Reactions

• Hydrolysis
• Acids or bases will hydrolyze the peptide
  bonds to give AAs.
• Denaturation
• Process in which proteins can lose their
  secondary, tertiary, quaternary structure,
  destroying their function, without breaking the
  peptide bonds.

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Denaturation of Proteins

• Disruption of any of the bonds that stabilize the
  secondary, tertiary, quaternary structure. The
  covalent amide bonds of the primary structure are
  not affected.
• Alters the shape of the protein and it becomes
  biologically inactive.
• Denaturing agents heat, acids, bases, organic
  compounds, heavy metal ions, mechanical agitation.

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Denaturation of Proteins

• Acids and bases disrupt salt bridges and hydrogen
  bonds
• Heat,UV,microwave,agitation disrupt hydrophobic
  bonds and hydrogen bonds
• Heavy metals like Hg2, Ag, Pb2 disrupt the
  disulfide bonds
• Organic molecules disrupt hydrophobic interactions

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Enzymes

• Enzymes are protein catalysts, most are globular
  proteins and are very specific. They increase the
  rate of the reaction by lowering the activation
  energy, and operate at mild temperature and pH.
  Reactants are called substrates.
• Classification
• Named according to the type of reaction and the
  substrate.
• 1. Oxidoreductases - biological oxidation and
  reduction reactions, e.g. lactate dehydrogenase.
• 2. Transferases - switch functional groups
  between molecules, e.g. transaminases.
• 3. Hydrolases - hydrolyze molecules, e.g.
  sucrase.
• 4. Lyases - remove groups without using water,
  break C-O, C-C, C-N bonds, e.g. pyruvate
  decarboxylase.
• 5. Isomerases - change one molecule into one of
  its isomers,(rearrange functional groups) e.g.
  phosphoglucose isomerase.
• 6. Ligases - form new bonds between C and N, O,
  S or another C, e.g. DNA polymerases.(join two
  molecules)

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Enzyme Constitution

• Can be pure protein, e.g. trypsin.
• Others are made up of a protein part called the
  apoenzyme and a nonprotein part called a
  cofactor, the combination is called a holoenzyme.
  The cofactor(s) can be ions (Mg2,Mn2,Ni2,
  Zn2,Fe2,Fe3,Cu2)or small organic molecules
  called coenzymes, these are frequently built from
  vitamins. (water soluble vitamins B C function
  as coenzymes)
• Some proteins are made in an inactive form called
  a zymogen or proenzyme, these must be activated
  by the active enzyme itself or some other enzyme,
  Examples are pepsinogen the proenzyme for pepsin
  and prochymotrypsin for chymotrypsin.

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Mechanism 1

• Active site, this is where the catalytic activity
  takes place.
• Functional groups in the active site interact
  with substrate(s) in such a way as to facilitate
  the making or breaking of bonds.
• The active site of the enzyme may be fixed,
  rigid, nonflexible shape, that fits the substrate
  exactly, this is the lock-and-key model by Emil
  Fisher 1890.
• The active site of the enzyme may conform itself
  to the substrate, the structure of the enzyme is
  flexible, this is the induced-fit model proposed
  by Daniel Koshland 1958.
• Different enzymes vary between the two extremes.

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Mechanism 2

• In both cases a reversible reaction with the
  substrate forms an enzyme-substrate complex, this
  then changes to release the product.
• E S ? E-S Then
• E-S ? E P
• Example
• Sucrase Sucrose ?Sucrase-sucrose complex
• Sucrase-sucrose complex?Sucrase Glucose
  Fructose

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Enzyme Specificity

• Enzymes will only work on one substrate or a few
  related molecules this is specificity.
• Absolutely - only one substrate, not common.
• Stereochemically - only work on one of two
  stereoisomers, D- or L- form.
• Group - work on different molecules containing
  the same functional group.
• Linkage - act on a particular kind of bond.

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Factors Affecting Enzyme Activity

• Substrate concentration
• Each enzyme molecule has a limit to how fast it
  works, the turnover number. As the amount
  concentration) of substrate is increased(while
  the enzyme concentration is kept constant) the
  rate will increase until each molecule is working
  at its maximal rate, then the activity is maximal
  and adding more substrate will not increase the
  rate any further.(when the enzyme gets saturated
  with substrate the rate reaches its maximum so it
  will level off, adding more substrate will not
  affect the rate anymore.)

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Factors Affecting Enzyme Activity

• Enzyme concentration
• With an unlimited amount of substrate, as more
  enzyme is added the rate increases in direct
  proportion this curve does not level off.
• Temperature
• As the temperature is raised initially the rate
  increases. Later the increased vibration within
  the molecule distort the active site and the rate
  increase slows down, reaches a maximum - the
  optimal temperature.
• Beyond this point the rate gets slower and
  finally stops when the enzyme is denatured.
• For most tissues the optimum temperature will be
  37oC, and it is the temperature at which an
  enzyme operates at maximum efficiency.
• (in the liver ca. 40oC).
• At lower temperatures enzymes show little
  activity and at higher than 50oC they lose their
  activity, they become inactive.

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Factors Affecting Enzyme Activity

• pH
• At the optimum pH an enzyme is most active
• At one particular pH value, known as the optimal
  pH7.4, the active site will be in the correct
  configuration, above or below that value ionic
  side groups may gain or lose charges. This will
  affect the enzyme structure and/or alter the
  active site, the shape, the solubility. This
  will cause the activity to decrease, eventually
  the enzyme will be denatured and activity will
  cease. For most tissues the pH will be ca. 7.4,
  stomach ca. 1-2 and small intestine ca. 8-9.

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Enzyme Inhibition

• Inhibitors---cause a loss of catalytic activity
  (release materials that block the active site of
  the enzyme toxins, drugs, metal complexes,
  substrate analogs).
• Irreversible---form covalent or very strong
  noncovalent bonds
• Reversible---form weak noncovalent bonds
• Inhibitors
• Competitive
• Noncompetitive

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Inhibitors

• Competitive has a structure that resembles the
  substrate and competes with the substrate for the
  active site
• Adding more substrate reverses the effect of the
  inhibitor
• Noncompetitive has a structure that does not
  resemble the substrate and it binds at a location
  other than the active site alters the 3-D
  structure of the enzyme so that a substrate
  cannot enter the active site
• Adding more substrate will not reverse the effect
  of the inhibitor
• Examples heavy metal ions Pb2, Ag, Hg2 that
  bond to COO- or OH
• Antibiotics produced by bacteria, mold, or yeast
  are inhibitors used to stop bacterial growth.

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Enzymes in Medicine

• Viral Hepatitis shown by presence in the blood
  of glutamatepyruvate aminotransferase (GPT) and
  glutamate oxalate aminotransferase (GOT).
• Myocardial Infarction shown by presence in the
  blood of creatine kinase (CK), GOT and lactate
  dehydrogenase (LD). Also LD has 5 isoenzymes LD1
  LD5. Normally the level of LD1 is lower than
  that of LD2, with a heart attack the ratio flips.
  

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Chemical reactions in the human body

• Every second thousands of chemical reactions
  occur in the cells of the human body.
• The purpose of the many chemical reactions in our
  bodies is to
• Store chemical energy in the body for future use
• Produce the essential amino acids
• Produce the essential lipids
• Release chemical energy for the production of
  macromolecules

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Nonpolar Amino Acids

• pI
• Glycine (gly or G) 6.00
• alanine (ala or A) CH3 - 6.11
• valine (val or V) (CH3)2CH- 5.96
• leucine (leu or L) (CH3)2CHCH2 - 5.98
• isoleucine (ile or I) CH3CH2CH(CH3)- 6.02
• phenylalanine (phe or F) Ph-CH2 - 5.48
• tryptophan (try or W) 5.89
• methionine (met or M) CH3-S-CH2CH2- 5.74
• proline (pro or P) 6.30

try
pro
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Polar Neutral

• pI
• serine (ser or S) HOCH2- 5.68
• threonine (thr or T) CH3CH(OH)- 5.64
• cysteine (cys or C) HSCH2- 5.07
• tyrosine (tyr or Y) HO-Ph-CH2- 5.66
• asparagine (asn or N) H2NCO-CH2 - 5.41
• glutamine (gln or Q) H2NCO-CH2CH2- 5.65

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Polar Acidic and Basic

• Polar, acidic
• aspartic acid (asp or D) HOOCCH2 - 2.77
• glutamic acid (glu or E) HOOCCH2CH2 - 3.22
• Polar, basic
• lysine (lys or K) H2NCH2CH2CH2CH2- 9.74
• histidine (his or H) 7.59
• Arginine 10.76