Download Bchem 4200 Part7 - U of L Class Index

Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 4200
II. Macromolecular Interactions
Introduction to Enzymes
What Characteristic Features Define Enzymes?
Enzymes are biochemical catalysts that have in common three
destinctive features:
1. catalytic power
2. regulation
3. specificity
Catalytic power can be defined as the ratio of enzyme-catalyzed rate
of a reaction to the unanalyzed rate.
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What Characteristic Features Define Enzymes?
Enzymes are biochemical catalysts that have in common three
destinctive features:
1. catalytic power
2. regulation
3. specificity
Regulation is achieved in a variety of ways:
amount of enzyme
reversible binding of inhibitors / activators
What Characteristic Features Define Enzymes?
Enzymes are biochemical catalysts that have in common three
destinctive features:
1. catalytic power
2. regulation
3. specificity
The selective qualities of enzymes are collectively recognized as its
specificity.
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Specific vs. Non-specific Binding
In order to carry out their function proteins have to specifically interact
with one or more “targets”:
Ions, small molecules, cofactors
nucleic acids, and other proteins.
What are the differences between specific and non-specific binding?
How can we distinguish between those two binding types?
Specific binding interactions are stoichiometric and saturate.
They usually have a high Ka for their substrate.
Specific vs. Non-specific Binding
The interaction of an enzyme and a “target” can be expressed as simple
Equilibrium for discrete binding sites or pockets.
-Single conformational product
-High affinity & discrimination
-Stoichiometric binding
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Equilibrium Constants
E + S ↔ ES
An association constant can be assigned:
Ka = Keq =
k1
k-1
[ES]
=
([E][S])
Affinities are usually expressed as dissociation constants, KD = 1 / Ka
High affinity typically nM
Low affinity typically mM
Example System: Ion Channels
Ion channels are integral membrane proteins that allow the passive transport
Of specific small ions in response to:
a) Electrochemical gradient
b) Transmembrane potential
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Example System: Ion Channels
Ion channels differ from transporters in at least three respects:
1) support very high rates of transfer (flux)
can approach diffusion limit (108 ions/s)
2) non-saturable rates of transfer
transfer rates increase as e.g. gradient
increases
3) They are gated and exist in at least
two states (open/closed)
Ion Channels - Basics
The driving force for a particular ion at given transmembrane potential (Vm)
can be calculated:
∆G = RT ln (
cinside
coutside
F = 96480 J/V
) + Z F Vm
R= 8.31 J/mol
2+
You have a model membrane system (at 22°C) with an ion channel for Ca .
2+
If the external Ca concentration is 10 mM, the internal is 0.1 mM and the
Transmembrane potential is -80 mV, what is the initial driving force for
Ca2+ transport?
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Ion Channels - Basics
You have a model membrane system (at 22°C) with an ion channel for Ca2+.
2+
If the external Ca concentration is 10 mM, the internal is 0.1 mM and the
Transmembrane potential is -80 mV, what is the initial driving force for
Ca2+ transport?
∆G = R T ln (cin / cout) + Z F Vm
= (8.31 J/mol K)(295 K) ln (0.1 mM/ 10 mM) + (2)(9648 J/mol V) (-80 mV)
= (-11296 J/mol) – (15440 J/mol)
= -26.74 kJ/mol
Water Channels
Aquaporins: Hydrophilic transmembrane channels for the passage
of water.
AQP1 homotetrameric integral membrane protein (e.g. red blood cells)
Water passes at high speed but protons are excluded efficiently.
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Aquaporin (AQP1)
AQP comprises 269
amino acid residues
Tandem repeat of
three transmembrane
α heices.
Loops B & E form
an aqueous pathway
Water flows at
9 -1
10 s through the
channel
Aquaporin (AQP1)
A transmembrane pore of 2-3 Å is formed
mainly due to conserved Asn192 and Asn76
Formation of a positive electrostatic field.
Approaching waters break their hydrogen bonds.
The interior of the channel is mainly hydrophobic
The pore allows passage a single file of water.
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Aquaporin (AQP1)
Why is it important to break the hydrogen bonding between the single water
molecules?
Aquaporin (AQP1)
X-ray structure: 1J4N (Bos taurus)
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Aquaporin (AQP1)
+
The Structure of the K Channel
Structure has been solved for Streptomyces lividans → prototype structure
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+
The Structure of the K Channel
Homotetramer
Each subunit is comprised of
two membrane spanning helices
and a short third helix.
Third helix is responsible for
ion selectivity.
+
Channels release K from the cell
+
These channels allow K (0.13 nm radius) to cross the membrane 10,000 x
+
more efficiently than Na (0.095 nm radius).
At a rate of about 108 ions/s !
+
Selectivity of the K Channel
1. The pore narrows near the exterior face of the membrane preventing ions
larger than K+ from exiting the cell.
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+
Selectivity of the K Channel
2. The pore is large on the cytosolic face and allows hydrated ions to enter.
+
Selectivity of the K Channel
2. The pore is large on the cytosolic face and allows hydrated ions to enter.
It narrows at the beginning of the selectivity helices, beeing smaller than
the hydrated Ions. Ions dehydrate in order to pass through the channel.
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+
Selectivity of the K Channel
3. Carbonyl oxygens (and helix diploes) of the selectivity helix mimic the
+
coordination shell of a single K . Energy costs for dehydration are small.
→ allows selectivity!
+
Additional Features of the K Channel
Neg. charged amino acidues attract cations.
Why is that?
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+
Additional Features of the K Channel
Two optimal K+ coordination sites 0.7 nm apart.
Why is that?
Additional Features of the Selectivity Filter
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Additional Features of the Selectivity Filter
View along the
channel from the
cytosolic side.
Summary from the Textbook
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+
Voltage-Gated Na Channel
Found along the length of axons and open when the transmembrane
+
potential drops (depolarization). Channel allows Na to enter the cell.
Channels open rapidly and than close as the transmembrane potential
drops. It remains closed for several ms before it can open again.
Voltage-gated sodium channels are highly specific for sodium (100 fold).
They are counteracted by voltage-gated K+ Channels.
+
Voltage-Gated Na Channel
Channel is formed by a large ~ 1850 AA long polyptide.
It contains 4 tandem repeats of of 6 transmembrane helices.
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+
Voltage-Gated Na Channel
+
Voltage-Gated Na Channel
The basic helix 4 moves in the membrane in response
to the local transmembrane potential.
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Acetylcholine Receptor
A low selectivity ligand-gated ion channel
Found in the postsynaptic membrane of neurons.
Opens in the presence of the neurotransmitter
2+
+
+
acetylcholine, allows the entry of Ca , K and Na .
Acetylcholine Receptor
All nAChR exist as heteropentamers.
The pore is about 20Å wide in parts of the channel
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Acetylcholine Receptor
Leu side chains prevent Ion influx.
Acetylcholine Receptor
Based on sequence similarities between different ligand-gated channels,
the γ-aminobutyric acid (GABA), glycin, and serotonin channels have been
classified to belong to the acetylcholine receptor superfamily.
→ probably similar 3D structure
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