Molecular Machinery

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Molecular Machinery

• Molecular basis of cell function
• Structure vs. Function
• Molecular mechanisms
• ENZYME ACTION
• Na K pump
• Cell Signalling

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ENZYMES

• Hydrolase hydrolysis
• Phosphatase REMOVE phosphate gps
• Protease break down proteins
• Nuclease break down nucleic acids
• ATPase hydrolyse ATP
• Kinase ADD phosphate gps
• Synthase join molecules
• Polymerase join molecules (to make
  polymers)
• Oxidoreductases electron transfer
• Isomerases isomerise p49-50 text

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How do Enzymes work?

• Lock key?

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Pyruvate Kinase Lock Key
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Carboxypeptidase
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glucose ATP à glucose-6-phosphate ADP
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INDUCED FIT THEORY OF ENZYME ACTION

• Example Hexokinase

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Active site

• 3 dimensional shape on the surface of the enzyme
• Contains specific amino acids to bind the
  substrate

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Induced Fit

• Substrate binding itself changes the shape of the
  protein
• INDUCED FIT

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Induced fit

• The change in the shape of the hexokinase has 2
  functions
• Changes shape of ATP binding site so it can bind
  ATP
• This prevents hexokinase acting independently as
  an ATPase
• Brings the two substrate binding sites closer,
  facilitating transfer of phosphate between the
  two molecules.

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CONTROL OF ENZYME ACTION

• Important not to waste valuable cell resources
• Prokaryotic cells
• enzyme synthesis is major control mechanism
• e.g. Jacob Monod Hypothesis of enzyme regulation
  (lac operon)
• Eukaryotic cells more complex

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CONTROL OF ENZYME ACTION in EUKARYOTICE CELLS

• Regulate transcription rate of enzyme gene same
  as Jacob Monod
• pH e.g. lysosomes pH 5
• Modify shape of protein itself
• Inhibitors
• Allosteric mechanisms
• Covalent modifications
• End-product inhibition

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Max reached because the active sites are full all
the time
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COMPETITIVE INHIBITORS

• Inhibitor binds (non covalently) to the active
  site
• Competes with substrate at active site
• Rate slows because active site encounters fewer
  substrate molecules per second.
• Competitive inhibitors have similar structure to
  the substrate
• Effect can be overcome by adding more substrate
  (increases chance of active site encountering
  substrate - competing out)

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Clinical application

• Methanol poisoning
• Methanol ? formaldehyde (toxic)
• Enzyme Alcohol dehydrogenase
• Formaldehyde Blindness Death (liver failure)
• Enzyme competitively inhibited by ethanol

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Non Competitive inhibitors

• Non Competitive Inhibitors (two types)
• Reversible bind non-covalently, reversibly to the
  enzyme
• Alter conformation of enzyme
• Not at the active site, not competed out by
  substrate
• e.g. inhibition of threonine deaminase by
  isoleucine, an example of end product inhibition
  ( allosteric modulation)

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Non Competitive Inhibitors

• Irreversible (could be on active site)
• Bind covalently, not able to be removed
• Alter conformation of enzyme
• Permanently inhibit enzyme
• e.g. Penicillins inhibit bacterial cell wall
  synthesis by non-competitivitvely binding to the
  enzymes
• Aspirin irreversibly binds to an enzyme which
  makes inflammatory lipids
• Organophosphorous inhibitors of
  Acetylchloinesterase

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ANTABUSE
DISULFIRAM or
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Allosteric Regulation

• Allosteric other (allo) steric (space/site)
• Some enzymes have alternative binding sites to
  which modulators (positive or negative non
  competitive inhibitor bind)
• They change the proteins shape.
• Allosteric enzymes often have multiple inhibitor
  or activator binding sites involved in switching
  between active and inactive shapes
• allows precise and responsive regulation of
  enzyme activity

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Cooperative substrate binding

• Allosteric or Regulatory enzymes can have
  multiple subunits (Quaternary Structure) and
  multiple active sites.
• Allosteric enzymes have active and inactive
  shapes differing in 3D structure.

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Sigmoid Reaction rate curves

• Enzymes with cooperative binding show a
  characteristic "S"-shaped curve for reaction rate
  vs.. substrate concentration. Why?
• Substrate binding is "cooperative."
• Binding of first substrate at first active site
  stimulates active shape, and promotes binding of
  second substrate.

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Covalent Modification

• Phosphorylation
• Phosphate added by kinases
• Removed by phosphatases

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Example Glycogen Phosphorylase

• Enzyme involved in breakdown of glycogen to
  produce glucose
• Inactive form not phosphorylated
• Active form phosphorylated
• Phosphorylase kinase adds phosphate groups (high
  energy needs)
• Phosphorylase phosphatase removes phosphate
  groups (low energy needs)
• Activity of phosphatase and kinase under hormonal
  control

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Allosteric modulation glycogen phosphorylase

• Glycogen phosphorylase also has binding sites for
  glucose, ATP AMP
• Glucose ATP indicate cell has a lot of energy
• Both negative allosteric modulators
• Adenosine Monophosphate (AMP formed by ATP
  hydrolysis) indicate cell has little energy
• Positive allosteric modulator

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Proteolytic Cleavage

• Proteolytic cleavage of a ZYMOGEN
• To produce active enzyme e.g.
• Trypsinogen ? trypsin
• Prolipase ? lipase
• Activation is required, otherwise these enzymes
  would digest the pancreas
• Pepsinogen ? pepsin
• This activation occurs through the action of
  pepsin itself
• Autocatalysis

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END PRODUCT INHIBITION

• Metabolic pathways usually involve a number of
  steps from precursor to product. e.g.

• Synthesis of isoleucine from threonine
• First step catalysed by threonine deaminase
• Allosterically inhibited by end product
  isoleucine
• Allows cell to monitor levels of product and
  control production rate appropriately

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NON COMPETITIVE INHIBITORS

• Bind to area of the protein other than the active
  site
• Alter conformation (shape) of the protein
  changing shape of active site/ making oit more
  difficult for substrate to bind
• Reversible non-covalently bound, can be diluted
  out
• Irreversible covalently bound cannot be diluted
  out

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• Induced fit Binding of glucose to Hexokinase
  induces a large conformational change (diagram p.
  386). The change in conformation brings the C6
  hydroxyl of glucose close to the terminal
  phosphate of ATP, and excludes water from the
  active site. This prevents the enzyme from
  catalyzing ATP hydrolysis, rather than transfer
  of phosphate to glucose. 
• It is a common motif for an enzyme active site to
  be located at an interface between protein
  domains that are connected by a flexible hinge
  region. The structural flexibility allows access
  to the active site, while permitting precise
  positioning of active site residues, and in some
  cases exclusion of water, following a
  substrate-induced conformational change.

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• This is a molecular model of the unbound
  carboxypeptidase A enzyme. The cpk, or
  space-filled, representation of atoms is used
  here to show the approximate volume and shape of
  the active site. Note the zinc ion (magenta) in
  the pocket of the active site. Three amino acids
  located near the active site (Arg 145, Tyr 248,
  and Glu 270) are labeled.
• This is a cpk representation of carboxypeptidase
  A with a substrate (turquoise) bound in the
  active site. The active site is in the induced
  conformation. The same three amino acids (Arg
  145, Tyr 248, and Glu 270) are labeled to
  demonstrate the shape change.