Getting to Know Enzymes Amylase

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Getting to Know Enzymes? - Amylase

• Chemistry II
• Mrs Shelley Baker

http//www.cryst.bbk.ac.uk/pps97/assignments/proje
cts/gazerro/amylase.gif
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Introduction to ? - Amylase

• ? - amylase enzymes belong to family 13 glycosyl
  hydrolases1.
• Catalyze the hydrolysis of ? - (1,4)- glycosidic
  linkages in starch.
• In humans, it is present in both salivary and
  pancreatic secretions.
• Human pancreatic ? - amylase is referred to as
  HPA2.

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? - Amylase

PDB ID1kbb Rydberg, E.H.,Li, C.,Maurus,
R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human
pancreatic alpha-amylase detailed kinetic and
structural studies of mutants of three conserved
carboxylic acids. Biochemistry v41
pp.4492-4502 , 2002 .
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Primary Structure of ? - Amylase

• 1 polypeptide chain comprised of 496 amino acid
  residues.
• The primary amino acid sequences of pancreatic
  and salivary ? - amylase are very homologous.
• Pancreatic and salivary ? - amylase share
  significant structural similarities3.

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Primary Structure of ? - Amylase
Backbone view
PDB ID1kbb Rydberg, E.H.,Li, C.,Maurus,
R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human
pancreatic alpha-amylase detailed kinetic and
structural studies of mutants of three conserved
carboxylic acids. Biochemistry v41
pp.4492-4502 , 2002 .
6
Secondary Structure of ? - Amylase

• 33 helical
• 20 helices, 165 residues.
• 18 beta sheet
• 21 strands, 90 residues.
• Remainder is extended strands

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Tertiary Structure of HPA

• 3 Structural Domains
• Domain A - Characteristic (?/?)8 barrel.
• Highly symmetrical fold of 8 parallel ?- strands
  arranged in a barrel encircled by 8 ?-helices1.
• (?/?)8 barrel first discovered in chicken muscle
  triose phosphate isomerase and is called the TIM
  barrel1.
• Characteristic of other enzymes in family 13.
• Site of catalysis and substrate binding1,3.
• Composed of residues 1-99 and 169-4043.

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Tertiary Structure of HPA

• Domain A - Characteristic (?/?)8 barrel.
• Location of substrate active site
• Active site residues Asp197, Glu233, and
  Asp300.
• Cl- binding site3.

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Tertiary Structure of HPA

• (?/?)8 barrel - center to lower left portion of
  the structure.
• Note strands are not shown.

PDB ID1kbb Rydberg, E.H.,Li, C.,Maurus,
R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human
pancreatic alpha-amylase detailed kinetic and
structural studies of mutants of three conserved
carboxylic acids. Biochemistry v41
pp.4492-4502 , 2002 .
10
Tertiary Structure of HPA

• Domain B
• Lies next to ?-barrel of domain A between ?-sheet
  3 and ?-helix 31.
• Forms a Ca2 binding site1,3.
• All ?-amylases are metalloenzymes and contain at
  least one Ca2 per enzyme molecule.
• Essential for activity and stability4.
• Composed of resideus 100-1683.

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Tertiary Structure of HPA

• Domain C
• Loosely associated with the other two domains.
• Function is unknown.
• Residues 405 - 496.
• Other members of the ? - amylase family contain a
  possible 6 other domains (D - I)3.

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Tertiary Structure of HPA

• Ball and stick view.
• Green, light blue and dark blue indicate Domains
  A, B, and C.

PDB ID1kbb Rydberg, E.H.,Li, C.,Maurus,
R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human
pancreatic alpha-amylase detailed kinetic and
structural studies of mutants of three conserved
carboxylic acids. Biochemistry v41
pp.4492-4502 , 2002 .
13
Note the locations of the Cl- site in the
?-barrel and Ca2 site in Domain B.
PDB ID1kbb Rydberg, E.H.,Li, C.,Maurus,
R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human
pancreatic alpha-amylase detailed kinetic and
structural studies of mutants of three conserved
carboxylic acids. Biochemistry v41
pp.4492-4502 , 2002 .
14
Note the relative positions of each structural
domain as well as the locations of the catalytic
amino acids, and the Ca2 and Cl- binding sites.
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Hydrophobic amino acid sequences of HPA
Primary structure- Hydrophobic sequences are
shown in red.
PDB ID1kbb Rydberg, E.H.,Li, C.,Maurus,
R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human
pancreatic alpha-amylase detailed kinetic and
structural studies of mutants of three conserved
carboxylic acids. Biochemistry v41
pp.4492-4502 , 2002 .
16
Hydrophilic amino acid sequences of HPA
Primary structure- Hydrophibic sequences are
shown in red. Note Asp197.
PDB ID1kbb Rydberg, E.H.,Li, C.,Maurus,
R.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Mechanistic analyses of catalysis in human
pancreatic alpha-amylase detailed kinetic and
structural studies of mutants of three conserved
carboxylic acids. Biochemistry v41
pp.4492-4502 , 2002 .
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Kyte-Doolittle Hydropathy Plot

• Hydrophobicity of an amino acid determines where
  the amino acid will be located in the final
  structure of the protein5.
• For globular proteins, the hydrophobic groups are
  located on the inside of the protein. Why?
• Where are the hydrophilic proteins? Why?

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Kyte-Doolittle Hydropathy Plot

• The structure of the protein defines its
  function.
• Kyte-Doolittle hydropathy plots
• Each amino acid is given a hydrophobicity score
  between 4.6 and -4.6 with 4.6 being most
  hydrophobic, -4.6 most hydrophilic.
• You set a window size - the number of amino acids
  whose hydrophobicity scores will be averaged6.

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Kyte-Doolittle Hydropathy Plot

• The computer program moves one amino acid and
  takes another average hydrophobicity score.
• This continues to the end of the protein.
• An average value is assigned to each amino acid.
  
• These averages are plotted on a graph6.

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Kyte-Doolittle Hydropathy Plot for HPA
Note The red line indicates the average
hydropathy score for all amino acids in the
protein.
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l?course_id_21585_1
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Kyte-Doolittle Hydropathy Plot for HPA

• Asp197, Glu233, and Asp300 are the three active
  site residues which are located in the (?/?)8
  barrel.
• Examining the Kyte-Doolittle plot, as well as the
  primary structure, you will notice that there is
  a regular pattern of alternating hydrophilic and
  hydrophobic regions of about 10 residues in
  length.
• Note that one of the most hydrophilic regions is
  around Asp300.

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Reaction Mechanism for ? - Amylase

• The action of the enzyme involves 3 amino acids
  clustered at the bottom of a V-shaped active site
  cleft of the ((?/?)8 barrel.
• Asp197 D197
• Gluc233 E233
• Asp300 D300 2

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Reaction Mechanism for ? - Amylase

• Asp aspartic acid
• Glu Glutamic acid

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Reaction Mechanism for ? - Amylase

• Acts on ? - glycosidic bonds of starch or
  glycogen (the substrates).
• Asp197 acts as the catalytic nucleophile.
• Glu233 acts as the acid/base catalyst.
• Asp300
• Aids in the deformation of the sugar.
• Plays a role in enhancing the electrophilicity of
  the sugar anomeric C by a H-bonding interaction
  with the hydroxyl group at C2.
• Above is based on kinetic and profile
  distribution data but the precise role is still
  unclear2.

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Reaction Mechanism for ? - Amylase

• Other amino acid residues such as histidine,
  argine and tyrosine may play a role in the
  following
• Positioning the substrate with the correct
  orientation in the active site.
• Proper orientation of the nucleophile.
• Transition state stabilization.
• Polarization of the electronic structure of the
  substrate1.

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Reaction Mechanism for ? - Amylase

• Step 1 Glu233 donates a proton to glycosidic
  bonded oxygen between 2 glucose molecules.

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Reaction Mechanism for ? - Amylase

• Nucleophilic Asp197 attacks the C1 of glucose.

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Transition state forms
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Reaction Mechanism for ? - Amylase

• Step 2 Release of the protonated glucose, a
  short polysaccharide fragment, or
  oligosaccharide.
• A new glucose molecule or water molecule moves
  into the active site.
• Forms a covalent intermediate.

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Reaction Mechanism for ? - Amylase
2
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Reaction Mechanism for ? - Amylase

• Step 3 The new molecule attacks the covalent
  bond between the glucose and the aspartic acid.
• A second transition state forms.

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Reaction Mechanism for ? - Amylase
Transition state
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Reaction Mechanism for ? - Amylase

• Glu233 accepts a hydrogen from the incoming
  species
• The oxygen of the incoming species replaces the
  oxocarbonium bond between the glucose molecule7.
• This mechanism was proposed by Koshland in 1953.

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Reaction Mechanism for ? - Amylase
Transition state
Final outcome
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Catalytic Parameters for ? - Amylase

• Vmax and KM (Michaelis constant) define the rate
  of the enzyme-catalyzed reaction.
• When S gtgt KM, then v Vmax.
• Velocity is no longer dependent on S so the
  reaction is obeying zero order kinetics. Every
  enzyme molecule has its enzyme-substrate binding
  site occupied by S8,9.

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Catalytic Parameters for ? - Amylase

• When Slt KM then v ? (vmax)S
• KM
• which means rate follows a first-order rate
  equation, v k?A where k? (vmax)
• KM
• KM for HPA and starch at optimal pH is 2.51mg per
  mL10.
• Optimal pH for enzymatic activity is 710.

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Catalytic Parameters for ? - Amylase

• kcat is a measure of an enzymes maximal
  catalytic activity.
• kcat number of substrate molecules converted
  into product per enzyme molecule per unit time
  when the enzyme is saturated with substrate8,9.

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Catalytic Parameters for HPA
3
Table cropped by writer to only include HPA data
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References
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References