Transcript De niet-covalente interacties

Lactate dehydrogenase
+ 38 ATP
+ 2 ATP
How does lactate dehydrogenase perform
its catalytic function ?
The reaction
Pyruvate + NADH + H+
Nicotinamide : absorbs
light at 340 nm and
causes fluorescence
NAD+ + Lactate
LDH
Transfer of a
hydride (= H )
Pyruvate
Lactate
-
H
Oxamate
+
H
• How does LDH bind NADH and pyruvate ?
• Where is the proton in the reaction coming from ?
• What determines the pH dependence of the catalytic reaction ?
• How does LDH decrease the activation energy of the reaction ?
• What determines the turnover rate of the enzyme ?
• Why is the NADH fluorescence increased when bound to LDH?
Properties of the enzyme are determined by
the properties and spatial arrangement of
amino acids in the 3D-structure of the
protein:
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Position
Size
Polarity
Interactions
Chemical properties
Subdivision of amino acids according to
chemical properties of their side chains
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Aliphatic: (Gly), Ala, Val, Leu, Ile, Met
Cyclic: Pro
Aromatic: Phe, Tyr, Trp
Sulfhydryl: Cys
Aliphatic hydroxygroup: Ser, Thr
Basic group: His, Lys, Arg
Carboxy group: Asp, Glu
Carboxamide: Asn, Gln
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Apolar-hydrophobic
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Polar-hydrophilic
http://nl.wikipedia.org/wiki/Aminozuur
The 3D-structure of a protein can be
determined by X-ray diffraction
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The protein is crystallyzed
The protein crystals are irratiated with X-rays
X-rays are diffracted by the atoms in the protein
From the diffraction pattern, the position of each atom in
the protein (except for H-atoms) is determined (electron
density map).
• The structure of the protein is reconstructed from the
electron density map, up to 0.2 Å accuracy. This gives
the exact position of each amino acid , substrates, etc. in
the 3D-structure
The 3D-structure of a protein can be
determined by X-ray diffraction
• The coordinates of each atom in the 3D-structure are
stored in a text file:
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
9
10
11
12
13
14
15
N
CA
C
O
CB
OG1
CG2
THR
THR
THR
THR
THR
THR
THR
2
2
2
2
2
2
2
-35.012
-33.877
-33.432
-33.479
-32.755
-33.084
-31.303
7.200
7.892
6.971
5.746
8.236
9.612
8.072
18.016
17.356
16.211
16.445
18.389
18.839
17.905
1.00
1.00
1.00
1.00
1.00
1.00
1.00
24.65
24.25
23.41
23.12
24.34
24.79
24.11
• For LDH, this text file is 104 pages long
6LDH
6LDH
6LDH
6LDH
6LDH
6LDH
330
331
332
333
334
335
The 3D-structure of a protein can be
determined by X-ray diffraction
• These coordinates can be read in a viewer to give a
‘picture’ of the protein:
What determines the turnover
rate of LDH ?
How do substrates bind ?
What interactions force the substrates in a position in which they can react ?
Non-covalent interactions in
proteins
• Electrostatic (or ionogenic) interactions
• Hydrogen bridges
• Vanderwaals interactions
• Hydrophobic interactions
Electrostatic interactions
The energy content of an electrostatic interaction
is described by:
E = (k*q1*q2) / (D*r)
In water (polar environment), with D = 80 en 3 Å, the
energy content is 5.8 kJ.mol-1 (1.4 kcal.mol-1)
In apolar, pure hexane (without water, D = 2), the
energy content is appr. 40x higher.
Hydrogen bridges (partial
ionogenic interactions)
• The strongest H-bond interactions are linear (donor – H
atom – acceptor).
• Hydrogen bridges have an energy content of 4 - 20
kJ.mol-1 (1 - 5 kcal.mol-1).
• The distance between N-H---O is less than between CH---O (3.5Ǻ).
Vanderwaals interactions
Vanderwaals interactions are
forces between permanent
and/or induced dipoles in
electrically neutral molecules.
Therefore, they behave like
partial ionogenic interactions
The energy content is 2 - 4
kJ.mol-1 (0.5 - 1 kcal.mol-1).
Hydrophobic interactions
• Association of apolar groups/molecules in water results in the release of
water molecules that surround the apolar surface in a stiff, ice-like structure.
• The released water molecules have more possibilities to interact with other
water molecules in solution.
• This results in an increase of the entropy (S) of the water in: G = H TS. This results in a decrease in the free energy. Hydrophobic interactions
are responsible for clustering of amino acids with hydrophobic residues in
the center of a protein.
pH determines if an amino acid is charged or
not, depending on the pKa
Why are the backbone NH2 and COOH-groups of amino acids not
important in proteins ? Which groups are important ?
Acid-base reactions
+
BH
B +
+
H
pKa = pH at which 50% is in the BH+ form and 50% is in de
B form.
Examples of aminoacid sidechains:
-COOH
-COO- + H+ ; pKa ~ 3.9
-NH3+
-NH2 + H+ ; pKa ~ 10.9
How is it possible to shift a pKa to a higher value ?
Free energy of a reaction
• Standard free energy of the reaction between pyruvate
and NADH is negative:
pyruvate + NADH + H+  lactate + NAD+; ΔG0pH 7.0 = - 6 kcal/mole.
• However, pyruvate and NADH do not react
spontaneously; this is because of the ‘activation energy’
• Enzymes decrease the activation energy of a reaction
The transition state of pyruvate
reduction by NADH
Amino acids in the protein stabilize the transition state of pyruvate reduction
by polarization of the carbonyl group