Coulomb interactions between internal ionizable groups and surface residues

1. Coulomb interactions between internal ionizable groups and surface residues Victor Khangulov May 13, 2009 Institute in Multiscale Modeling of Biological interactions Johns Hopkins University Laboratory of Dr. Garcia-Moreno

2. Many biochemical processes are governed by internal ionizable groups • Photoactivation • Ion homeostasis • H+ transport • e- transfer • Catalysis Most ionizable residues are located on the protein surface. Internal ionizable residues are responsible all forms of energy transduction:

3. Many industrial processes require altered pH dependent properties of enzymes • Starch liquefaction (production of ethanol and high-fructose syrup) • α-amylase is not active below pH 6. • Addition of salts is needed to adjust its activity to lower pH. [Andrew Shaw, Richard Bott, and Anthony Day Current Opinion in Biotechnology 10 (4), 349 (1999)] • Dye bleaching/Detergents • Fungal peroxidase is not active at higher pH (~10) and high peroxide concentration. • Directed evolution is necessary to increase its activity. [Joel Cherry et al. Nature biotechnology 17 (4), 379-84 (Apr 1999) ] • Feed additives (digestion of phosphorous as phytate) • Phytase: very low activity at lower pH (3.5) of the stomach. [Taewan Kim et al. Applied and Environmental Microbiology 72 (6), 4397 (2006)] Interactions between surface and active site ionizable groups can be modified to change pH profile of enzyme activity.

4. Acetoacetate decarboxylase – catalyzes formation of acetone and CO2 from acetoacetate. Mandelate racemase – catalyzes equilibration of (R)- and (S)-enaintioners of mandelate. Internal lysines are crucial in catalysis Lys-115 pKa = 6.0 Lys-166 pKa = 6.4 Lane Highbarger and John Gerlt Biochemistry 35 (1), 41 (1996 Bharati Mitra et al. Biochemistry 34 (9), 2777 (1995)

5. Apparent pKa values of Lys at 25 internal positions Normal pKa of Lys in water

6. pKa Determination: Ideal CaseTitration of Lys-25 inL25K Background L25K • Assumption: Group behaves independently • We know pKa values of all H,D, and E! • His-8 pKa = 6.3 • His-121 pKa = 5.4 • Asp-21 pKa = 6.5 • Everything else titrates ≤ 4.5 • For “ideal” cases, H8, H121 and D21 are not affected by internal lysine.

7. pKa Determination: Non-Ideal CaseTitration of Lys-62 inT62K Background T62K His-8, His-121 or Asp-21 are Affected by the ionization of Lys-62

8. pKa of Lys-62 shifts down in D21N variant Background Background T62K D21N/T62K pKa = 7.0 ± 0.2 pKa = 8.1 ± 0.1

9. Titration of Asp-21 shows dependence on the presence of Lys-62 Asp-21 in T62K pKa ≈ 4.3 ± 0.5 n = 0.6 ± 0.03 Asp-21 in ∆+PHS pKa = 6.6 ± 0.1 n = 2.0 ± 0.02

10. Titration of other groups in the presence of Lys-62 T62K T62K ∆+PHS ∆+PHS D19 D21 T62K ∆+PHS ∆+PHS T62K E67 E43

11. Effect of Lys-62 on the pKa of Asp-21 T62K ∆+PHS pKa1 = 4.3 pKa2 = 6.6 D21N D21N/T62K ∆Gij = 1.36 (pKa2 – pKa1) = 1.36 (6.6 – 4.3) = 3.0 kcal/mol

12. NMR confirms pKa of Lys-62 obtained through linkage analysis T62K pKa = 8.1 ± 0.02 (Linkage pKa = 8.1 ± 0.1) Global fit of 1H amide chemical shift

13. Lys-62 pKa shifts further down in D21N variant D21N/T62K pKa = 6.7 ± 0.03 (Linkage pKa = 7.0 ± 0.2)

14. Effect of Asp-21 on pKa of Lys-62 T62K ∆+PHS pKa2 = 8.1 pKa1 = 6.7 D21N D21N/T62K ∆Gij = 1.36 (pKa2 – pKa1) = 1.36 (8.1 – 6.7 ) = 1.9 kcal/mol

15. ∆Gij is not symmetric between Asp-21 and Lys-62 T62K ∆+PHS pKa2 = 8.1 pKa1 = 4.3 pKa2 = 6.6 This is the best estimate of ∆Gij pKa1 = 6.7 D21N D21N/T62K ∆Gij (Lys-62) = 1.9 kcal/mol ∆Gij (Asp-21) = 3.0 kcal/mol

16. Electronic polarization Bulk water Fixed permanent dipoles Water penetration Relaxation of permanent dipoles Second internal charge Surface charges Local unfolding Fluctuations of surface charges Global unfolding The magnitude of the coupling between internal and surface ionizable groups could be governed by other factors

17. T62K structure with Lys-62 in neutral state Background T62K

18. Isolated regions exhibit large changes in 1HN chemical shift upon titration of Lys-62 (pH 7-9) Asp-21 region Lys-62 region

19. Summary • There appears to be significant interaction between Lys-62 and Asp-21. • Titration of Lys-62 does not induce major structural reorganizations. • Ionization of Lys-62 induces minor structural reorganizations. • The Coulomb interaction between Asp-21 and Lys-62 is 1.9 kcal/mol.

20. 9 Internal Lys Variants Show Evidence of Coupling to another ionizable Residue T62K

21. Crystal structures where internal Lys is coupled to the ionization of Asp-21 6 Å 9 Å T62K V104K

22. Future experiments: • What are the structural consequences of ionization of Lys-62? • Is the phenomenon observed in T62K general? (study 8 Lys variants that exhibit coupling to another residue).

23. Progress on x-ray structures

24. His-8 and His-121 pKa values show no dependence on the presence of Lys-62

25. Simulation of interactions between two ionizable groups Asp-21 pKa = 5.0 Lys-62 pKa = 8.1 Asp-21 pKa = 6.5 Lys-36 pKa = 7.2

26. + Relationship between Gibbs free energy and a shift in pKa value

27. The free energy of ionization of an internal group can be calculated from its pKa shift

28. Primary energetic contributions to the pKa value of internal groups Always unfavorable pKa of Glu  If unfavorable, pKa of Glu  If favorable, pKa of Glu 

29. Modified Hill Equation 2-site 1-site

30. Surface mutations affect enzyme catalysis • Serine protease Russell, A. J., and Fersht, A. R. (1987) Nature328(6130), 496-500 Jackson, S. E., and Fersht, A. R. (1993) Biochemistry32(50), 13909-13916 • Catalytic His-64 interacts with surface Asp-99 (13 Å away) and Glu-156 (15 Å away) • ∆pKa ≈ 0.4, ∆G ≈ 0.6 kcal/mol • Thermolysin-like protease de Kreij, A., van den Burg, B., Venema, G., Vriend, G., Eijsink, V. G., and Nielsen, J. E. (2002) J Biol Chem277(18), 15432-15438 • Catalytic Glu-143 and His-231 interacts with carboxylic groups 10-15 Å away. • Max ∆pKa ≈ 0.5, ∆G ≈ 0.6 kcal/mol