115317 Industrial Enzymes

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115317 Industrial Enzymes

• ?.??????? ??????
• ??????????????????????
• ?????????????? ???????????????? ??????????????
• ?????????????????

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Enzymes are important

• ???????????????????????????
• ??????????????????????????????????????????????
• Galactose metabolism, glucose metabolism
• ?????????????????????????????????????????????
  ???? amino acids, lipids
• ?????????????????????
• Hydrolysis of proteins, carbohydrates and fats
• Health and Disease
• ??????????????????????????????????? 400 ????

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Enzyme and diseases

• Lactose intolerance
• ?-galactosidase
• Lactose
  Galactose Glucose
• (not taken up) (taken up by intestine
• 
  mucosa)

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Enzymes are Tools

• For analysis (Methods of Enzymic Analysis,
  Bergemeyer)
• Colorimetric assays (coupled enzyme assays)
• Enzyme electrodes
• As indicators (e.g. ELISA)
• Biotransformation
• Molecular biology
• Reactors, immobilized enzymes
• Food Processing

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Food Processing

• Clot milk for cheese (chymosin)
• Hydrolyze starch for corn sweetener (amylase,
  glucose isomerase)

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Industrial uses of enzymes
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Enzymes in cloning

• Purpose
• Polymerase chain reaction
• DNA from RNA
• Sequence-specific cuts
• Removing phosphate
• Splicing
• Selectable markers

• Enzymes
• Heat-stable polymerase
• Reverse transcriptase
• Restriction endonucleases
• Phosphatase
• Ligase
• Antibiotic resistance

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Enzyme are Catalysts

• Increase the rate of chemical reactions
• Other catalysts that are not enzymes platinum,
  palladium, H, OH-

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This class covers

• Part I
• Enzyme structure
• Properties of enzymes/proteins
  chemical/physical properties, separation and
  purification
• Kinetics and Mechanisms
• Classification
• Part II
• Enzyme applications

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Enzyme Structure
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????????????????????

• ???????????????? (Primary structure)
• sequence
• ?????????????????? (Secondary structure)
• repeating structural patterns defined by ??and ?
• 3. ????????????????? (Tertiary structure)
• overall fold
• 4. ????????????????? (Quaternary structure)
• subunit structure

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Protein are Linear Polymer of Amino Acids
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Small, Neutral Amino Acids
Glycine G 7.2 occurrence Alanine A 7.2
occurrence
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Polar, Uncharged Amino Acids
Serine S 6.8
Cysteine C 1.9
Threonine T 5.9
Methionine M 2.2
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More Polar Amino Acids
Histidine H 2.3 Asparagine
N 4.3 Glutamine Q 4.3
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Acidic Amino AcidsAnionic at Neutral pH
Aspartate D 5.3 Glutamate E 6.3
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Basic Amino AcidsCationic at Neutral pH
Lysine K 5.9 Arginine R 5.1
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Hydrophobic Amino Acids
Valine V 6.6 Leucine L 9.1 Isoleucine
I 5.3
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Aromatic Amino Acids
Phenylalanine F 3.9 Tyrosine Y 3.2 Tryptop
han W 1.4
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The Imino Acid
Proline P 5.2
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????????????????????

• ???????????????? (Primary structure)
• sequence
• ?????????????????? (Secondary structure)
• repeating structural patterns defined by ??and ?
• 3. ????????????????? (Tertiary structure)
• overall fold
• 4. ????????????????? (Quaternary structure)
• subunit structure

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Amino Acids and Peptide Bonds
????????????? polymers ???????????????????
(one-dimensional), ??????? (fold)
???????????????????????? (three-dimensional
structures)
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???????????? Peptide Bonds
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More F Y
Another Depiction of Torsion Angles ??and ?
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Phi and Psi Angles
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Secondary structure

• ?? 2 ??? ????
• - Helix
• ? - Sheet

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HydrogenBondsbetweenwater and other donors
andacceptors
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? - Helix
3.6 residues per turn 5.4 Å per turn
("pitch") 1.5Å per residue ("rise"), 18 residues
27Å
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(No Transcript)
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Periodicity Along the HelixA Helical Wheel
360 per turn 3.6 residues/turn 100/residue
Shiffer Edmundson
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? - Sheet
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Antiparallel ? - Sheet

• ??? ?- strands ????????? run ?????????????????????
  Hydrogen bonds ??????? NH ??? CO groups
  ??????????????????????????? strand ???????????
  stabilizing ?????????

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Parallel ? - Sheet

• ??? ?- strands ????????? run ????????????????

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Ramachandran Diagram
180
y (degrees)
0
-180
0
180
-180
f (degrees)
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Secondary Structure and Ramachandran's Plot
180
Polyproline Helix
Collagen Helix
b Strand in antiparallel sheet
Type II turn
b Strand in parallel sheet
a Helix (left)
y (degrees)
0
310 Helix
Type II turn
a Helix
Extended Chain
-180
0
180
-180
f (degrees)
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Beta Turns
Features 4-residue turns COi
often H-bonded to NHi3
i
i1
i2
i3
i
i1
i2
i3
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Gamma Turns
Features 3-residue turns COi often H-bonded
to NHi2
i
i1
i2
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Summary of 2o Structure

• ?-helix
• ?-strand
• tight turns
• ?-turns
• ?-turns
• random coil

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Protein Folding Domains
Tertiary structure
A domain is a portion of a protein that folds
independently of other regions of the protein
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Amino Acids and Peptide Bonds
????????????? polymers ???????????????????
(one-dimensional), ??????? (fold)
???????????????????????? (three-dimensional
structures) ????????????????????????? Hydrophobi
c effect Disulfide bridges Salt bridges Hydrogen
bonding
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Hydrogen Bond betweenTwo Water Molecules
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It is not so much hydrophobicity,but
insufficient hydrophilicityto pry the water
molecules apart...
C. Chothia
A hydrophobic solute forces the water to orient
itself to maximize H-bonding. Entropy is lower.
The solute should present the smallest surface
possible.
Water can H-bond extensively without regard to
orientation. There is always another water
molecule to H-bond to. Enthalpy is low and
entropy is high.
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Alcohol Dehydrogenasewith bound water in a
crystal
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Disulfide Bond Formation
Cys-CH2-SH HS-CH2-Cys
Cys-CH2-S-S-CH2-Cys

2H 2e-
(dont forget these)
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Disulfide Bonds in Lysozyme
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Salt Bridge Formation
???????????? an ionic bond ??????? charged
side-chain groups (e.g., Glu and
Lys). ???????????????????????????????? ionic
strength ??? salt bridge ?????????????????????????
??? (surface) ??????????????????????????????
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Salt bridge
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Strengths of Interactions(kcal/mole)
Peptide bond 120 Disulfide bond
80 electrostatic interaction 10-20 H-bonds
1-5 van der Waals attraction 3-5 other dipole
attraction weaker
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Summary of 2o Structure

• ?-helix
• ?-strand
• tight turns
• ?-turns
• ?-turns
• random coil

• Not too many ways proteins fold
• Folds can be put into four classes

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Classes of Protein Structures(C. Chothia)
? mostly ?-helix e.g., four-helix
bundle ? mostly ?-sheet e.g., ?-barrels ?/? repe
ating, alternating helix-sheet e.g.,
???-barrels ?? segregated helix and sheet
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A Mostly-Alpha Protein(the Globin Fold)
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The Four-Helix Bundle
20o
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Helix Stacking
50o
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Hairpin Turns and Antiparallel Sheet
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A ?-Barrel-adjacent ?-strandsare linked by
turns
?-meander
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A Jelly Roll Barrel
Barrel is formed from non-sequential strands. It
is closed at the top and bottom.
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The ????? motif
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??? Structure - the ???-Barrel
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??? Structure - the Twisted Sheet
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?-Structures - the antiparallel Sheet
A Greek Key motif
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Correlation between Fold and Function

• Essentially non
• Proteins with similar folds may have completely
  unrelated functions (e.g. flavodoxin and
  adenylate kinase)
• The fold provides a stable structure in which to
  embed an active site
• However, similar sequence implies similar fold
  and similar function
• The details of the active site determine
  specificity

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Amino AcidsProperties of the side chains
residue pKa H (K/D) residue pKa H
(K/D) Ile I ---- 4.5 Trp W ---- -0.9 Val V ----
4.2 Tyr Y 10.07 -1.3 Leu L ---- 3.8 Pro P ----
-1.6 Phe F ---- 2.8 His H 6.0 -3.2 Cys C 8.33
2.5 Asn N ---- -3.5 Met M ---- 1.9 Gln Q ----
-3.5 Ala A ---- 1.8 Asp D 3.86 -3.5 Gly G ----
-0.4 Glu E 4.25 -3.5 Thr T ---- -0.7 Lys K 10.
53 -3.9 Ser S ---- -0.8 Arg R 12.48 -4.5
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The Active Site

• The active site is the binding site for
  substrates and cofactors
• It is in a pocket in the enzyme
• The pocket buried inside the enzyme
• But it is accessible from the surface
• The structure may change upon binding of one or
  more substrates
• Some components (not reactants) may bind
  elsewhere and may affect activity

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Alcohol Dehydrogenase with bound substrates
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Ferric Horseradish Peroxidase
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Complex between pectin methylesterase and its
inhibitor protein
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Oligomeric Proteins (Quaternary structure)
Subunit-Subunit Interactions

• the hydrophobic effect
• hydrogen bonding (?-sheet)
• helix stacking
• salt bridges
• disulfide bonds
• metal ions

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Two Identical Subunits, Three Identical Subunits
How likely is this one?
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C2 Symmetry
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Horse ADH
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Three-Fold Symmetry
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Four Identical Subunits
Doesnt this imply chain formation?
What if subunits are not identical?
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Five-Fold Symmetry
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Five Identical Subunits
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Six Identical Subunits
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A Hexamer
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Glutamine Synthetase (dodecamer)
1994
ca. 1970