Transcript Cellular Mechanisms

Molecular Machinery
• Molecular basis of cell function
– Structure vs. Function
– Molecular mechanisms
• ENZYME ACTION
• Na+ K+ pump
• Cell Signalling
ENZYMES
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Hydrolase
Phosphatase
Protease
Nuclease
ATPase
hydrolysis
REMOVE phosphate gps
break down proteins
break down nucleic acids
hydrolyse ATP
• Kinase
• Synthase
• Polymerase
ADD phosphate gps
• Oxidoreductases
• Isomerases
p49-50 text
electron transfer
isomerise
join molecules
join molecules (to make
polymers)
How do Enzymes work?
• Lock & key?
Pyruvate Kinase Lock & Key
Carboxypeptidase
glucose + ATP  glucose-6-phosphate + ADP
INDUCED FIT THEORY OF ENZYME ACTION
• Example Hexokinase
Active site
• 3 dimensional shape
on the surface of the
enzyme
• Contains specific
amino acids to bind
the substrate
Induced Fit
• Substrate binding
itself changes the
shape of the protein
• INDUCED FIT
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.
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
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
Max reached because the active sites are full all the time
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)
Clinical application
• Methanol poisoning
– Methanol

formaldehyde (toxic)
– Enzyme Alcohol dehydrogenase
• Formaldehyde = Blindness & Death (liver failure)
– Enzyme competitively inhibited by ethanol
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)
Non Competitive Inhibitors
– Irreversible (could be on active site)
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Bind covalently, not able to be removed
Alter conformation of enzyme
Permanently inhibit enzyme
e.g. Penicillins inhibit bacterial cell wall synthesis by noncompetitivitvely binding to the enzymes
• Aspirin irreversibly binds to an enzyme which makes
inflammatory lipids
• Organophosphorous inhibitors of Acetylchloinesterase
ANTABUSE
DISULFIRAM or
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 protein’s 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
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.
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.
Covalent Modification
• Phosphorylation
– Phosphate added by kinases
– Removed by phosphatases
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
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
Proteolytic Cleavage
• Proteolytic cleavage of a ZYMOGEN
– To produce active enzyme e.g.
– Trypsinogen  trypsin
– Prolipase  lipase
• Activation is required, otherwise
Protease these
enzymes would digest the pancreas
– Pepsinogen  pepsin
– This activation occurs through the action of pepsin
itself
– Autocatalysis
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
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
• 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.
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• 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.
• 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