Lactate dehydrogenase + 38 ATP + 2 ATP

How does lactate dehydrogenase perform its catalytic function ?

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

Pyruvate Oxamate Lactate H-H- H+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: Position Size Polarity Interactions Chemical properties

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

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

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 LDH 330 ATOM 10 CA THR LDH 331 ATOM 11 C THR LDH 332 ATOM 12 O THR LDH 333 ATOM 13 CB THR LDH 334 ATOM 14 OG1 THR LDH 335 ATOM 15 CG2 THR For LDH, this text file is 104 pages long

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 kJ.mol -1 (1 - 5 kcal.mol -1 ). The distance between N-H---O is less than between C- H---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 kJ.mol -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 pK a Why are the backbone NH 2 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 -NH 3 + -NH 2 + 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 + ; ΔG 0 pH 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