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Lactate dehydrogenase

Lactate dehydrogenase. + 38 ATP. + 2 ATP. How does lactate dehydrogenase perform its catalytic function ?. Pyruvate + NADH + H + NAD + + Lactate. The reaction. LDH. Nicotinamide : absorbs light at 340 nm and causes fluorescence. Transfer of a hydride (= H - ).

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Lactate dehydrogenase

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  1. Lactate dehydrogenase + 38 ATP + 2 ATP

  2. How does lactate dehydrogenase perform its catalytic function ?

  3. Pyruvate + NADH + H+ NAD+ + Lactate The reaction LDH Nicotinamide : absorbs light at 340 nm and causes fluorescence Transfer of a hydride (= H-)

  4. Pyruvate Lactate H- H+ Oxamate

  5. 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?

  6. Properties of the enzyme are determined by the properties and spatial arrangement of amino acids in the 3D-structure of the protein: • Position • Size • Polarity • Interactions • Chemical properties

  7. Subdivision of amino acids according to chemical properties of their side chains • 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 • Apolar-hydrophobic • Polar-hydrophilic http://nl.wikipedia.org/wiki/Aminozuur

  8. The 3D-structure of a protein can be determined by X-ray diffraction • 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

  9. 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 9 N THR 2 -35.012 7.200 18.016 1.00 24.65 6LDH 330ATOM 10 CA THR 2 -33.877 7.892 17.356 1.00 24.25 6LDH 331ATOM 11 C THR 2 -33.432 6.971 16.211 1.00 23.41 6LDH 332ATOM 12 O THR 2 -33.479 5.746 16.445 1.00 23.12 6LDH 333ATOM 13 CB THR 2 -32.755 8.236 18.389 1.00 24.34 6LDH 334ATOM 14 OG1 THR 2 -33.084 9.612 18.839 1.00 24.79 6LDH 335ATOM 15 CG2 THR 2 -31.303 8.072 17.905 1.00 24.11 • For LDH, this text file is 104 pages long

  10. 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:

  11. What determines the turnover rate of LDH ?

  12. How do substrates bind ? What interactions force the substrates in a position in which they can react ?

  13. Non-covalent interactions in proteins • Electrostatic (or ionogenic) interactions • Hydrogen bridges • Vanderwaals interactions • Hydrophobic interactions

  14. 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.

  15. 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 C-H---O (3.5Ǻ).

  16. 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).

  17. 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.

  18. 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 ?

  19. 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 ?

  20. 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

  21. 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

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