Amnon Kohen Department of Chemistry The University of Iowa

1. Protein dynamics and tunneling effects in the DHFR and TS catalysis Amnon Kohen Department of Chemistry The University of Iowa

2. Overview • Background and experimental tools • Dihydrofolate Reductase (DHFR) • Dynamics-activity relationship • Thymidylate Synthase (TS) • Alternative TS (FDTS)

3. E R.C. A C + + B D Uncatalyzed reaction

4. Uncatalyzed vs. Enzyme-catalyzed reactions E R.C.

5. E R.C. Kinetic complexity

6. light isotope Tunneling of a bound particleGround-State Nuclear Tunneling

7. Size H [product] D time Temperature dependency AH/AT AD/AT 1.6 1.2 0.6 0.9 KIEs as Probe of Tunneling • Swain, C. G. et al., J. Am. Chem. Soc.1958, 80, 5885-5893 • Huskey, W. P.; Schowen, R. L. J. Am. Chem. Soc.1983, 105, 5704-5706. • Saunders, W. H. J. Am. Chem. Soc.1985, 107, 164-169. • Kohen, A.* and Jensen J.H. J. Am. Chem. Soc. 2002,124, 3858-3864. • Kohen, A.*Prog. React. Kin. Mech.2003, 28, 119-156.

8. AH/AT AD/AT 1.6 1.2 0.6 0.9 KIE Arrhenius Plots

9. Thymine biosynthesis

10. Overview • Background and experimental tools • Dihydrofolate Reductase (DHFR) • Dynamics-activity relationship • Thymidylate Synthase (TS) • Alternative TS (FDTS) Movie by Sawaya, M. R. and Kraut, J. Biochemistry 1997, 36, 586-603.

12. Dihydrofolate Reductase Radenine dinucleotide 2'-P R'(p-aminobenzoyl)glutamate

13. DHFR Kinetics Fierke et al. Biochemistry (1987) 26, 4085-4092

14. O O O O H T D T T D T H O O D D H H N H N H N H N H 2 2 2 2 N H N H 2 2 N N N N N N = = = R O P O = R O P O R O P O R O P O 3 3 3 3 = = R O P O R O P O * * 4 S - [ H , T ] - N A D P H 3 3 4 S - [ D , T ] - N A D P H 4 R - [ D , T ] - N A D P H 4 R - [ H , T ] - N A D P H 1 4 1 4 [ A d - C ] N A D P H [ A d - C ] N A D P H Competitive KIE experiments with DHFRMixed-labeled NADPH H/T KIE D/T KIE

15. GDH Glucose-1-D GDH Glucose-1-T GDH Glucose-1-H O O O O H T D T T D T H N H N H N H N H 2 2 2 2 N N N N = = = R O P O = R O P O R O P O R O P O 3 3 3 3 4 S - [ H , T ] - N A D P H 4 S - [ D , T ] - N A D P H 4 R - [ D , T ] - N A D P H 4 R - [ H , T ] - N A D P H Synthesis of Different Labeling Patterns for theC4 Position of Nicotinamide Ring

16. GDH glucose-1-D GDH glucose-1-H O O D D H H N H N H 2 2 N N = = R O P O R O P O * * 3 3 1 4 1 4 [ A d - C ] N A D P H [ A d - C ] N A D P H Synthesis of [Ad-14C;C4-2H2] and [Ad-14C;C4-1H2] NADPH

17. O O O O H T D T T D T H O O D D H H N H N H N H N H 2 2 2 2 N H N H 2 2 N N N N N N = = = R O P O = R O P O R O P O R O P O 3 3 3 3 = = R O P O R O P O * * 4 S - [ H , T ] - N A D P H 3 3 4 S - [ D , T ] - N A D P H 4 R - [ D , T ] - N A D P H 4 R - [ H , T ] - N A D P H 1 4 1 4 [ A d - C ] N A D P H [ A d - C ] N A D P H Competitive KIE experiments with DHFRMixed-labeled NADPH H/T KIE D/T KIE • Markham et al., (2003) Anal. Biochem.322, 26-32. • Agrawal, N., and Kohen, A. (2003) Anal. Biochem.322, 179-184 • Markham et al., (2004) Anal. Biochem., 325, 62-67. • McCracken et al., (2003) Anal. Biochem., 324, 131-136.

18. Determination of KIE Fractional conversion determination: Rt and R∞ determination: for any time point (t) to (∞) NADPH NADP+ H4F NADP+ NADPH

19. Coupled 1˚-2˚ motion From the mixed labeling experiment Ln(1.19)/ln(1.052)=3.4 ±1 —No coupled motion Calculated vs. experimental 2˚ H/D KIEs Calculated : 1.13 Mireia Garcia-Viloca, Donald G. Truhlar,* and Jiali Gao* Biochemistry 2003, 42, 13558-13575 Experimental: 1.13 ± 0.02 Equilibrium: 1.127 ± 0.009 Location of the transition state?

21. Extracting intrinsic KIE from H/D/T H/D/T data allow calculations of an intrinsic KIE: Northrop, D.B. In Enzyme mechanism from isotope effects; Cook, P. F., Ed.; CRC Press: Boca Raton, Fl., 1991, pp 181-202. http://cricket.chem.uiowa.edu/~kohen/tools.html

27. Al/Ah Upper Limits Al/Ah* H/D 3.50.5 1.4 H/T 7.01.5 1.6 D/T 1.700.14 1.2 Temperature Dependence as a Criterion for Tunneling Schneider & Stern (1972) J.A.C.S., 94, 1517-1522. Stern & Weston, (1974) J.Chem. Phys.., 60, 2815-2821. Bell (1980) The Tunneling Effect in Chemistry, Chapman & Hall, ED., London & New York. Melander & Saunders (1987) Reactions Rates of Isotopic Molecules, Krieger, Ed., Fl. Sikorski, R. S., Wang, L., Markham, K. A., Rajagopalan, P. T. R., Benkovic, S. J., and Kohen, A.* J. Am. Chem. Soc., 126, 4778-4779 (2004).

28. AH/AT AD/AT 1.6 1.2 0.6 0.9 KIE Arrhenius Plots

29. DHFR: Activation ParametersInitial velocity of kcat at pH = 9

30. Overview • Background and experimental tools • Dihydrofolate Reductase (DHFR) • Dynamics-activity relationship • Thymidylate Synthase (TS) • Alternative TS (FDTS)

31. Tunneling & Dynamics in theoretical modelsMarcus-like model of ground-state tunneling

32. Vibrationally Enhanced Tunneling

33. Diagram of a portion of the network of coupled promoting motions in DHFR. The yellow arrows and arc indicate the coupled promoting motions. Benkovic, Hammes-Shiffer and co-workers PNAS (2002) 99, 2794-2799.

34. MD calculations with DHFR Jennifer L. Radkiewicz and Charles L. Brooks, III* J. Am. Chem. Soc. 2000, 122, 225-231. (a) DHFR/DHF/NADPH Figure 5. Residue-residue based map of correlated motions. Red and yellow indicate regions of positive correlation, and dark blue indicates regions of anti-correlation.

35. MD calculations with DHFR Jennifer L. Radkiewicz and Charles L. Brooks, III* J. Am. Chem. Soc. 2000, 122, 225-231. (a) DHFR/DHF/NADPH (b) DHFR/THF/NADP+ Figure 5. Residue-residue based map of correlated motions. Red and yellow indicate regions of positive correlation, and dark blue indicates regions of anti-correlation.

36. MD calculations with DHFR Jennifer L. Radkiewicz and Charles L. Brooks, III* J. Am. Chem. Soc. 2000, 122, 225-231. (a) DHFR/DHF/NADPH (b) DHFR/THF/NADP+ (c) DHFR/THF/NADPH Figure 5. Residue-residue based map of correlated motions. Red and yellow indicate regions of positive correlation, and dark blue indicates regions of anti-correlation.

37. Dihydrofolate Reductase Agarwal et al., PNAS 2002, 99, 2794-2799.

38. DHFR Temperature Dependency - w.t. vs. G121V Commitment 3.2±0.3 H 3.7±0.2 Ea in kcal/mol At high and low temperature 7.3±0.5 H 11.9±0.5 D D 2.5±1.2 3.1±0.4 9.2±2.1 7.5±0.7 G121V: Wild Type: Intrinsic KIEs Observed KIEs Observed H/D on kcat Observed H/D on kcat Pre-steady-state KIE Intrinsic KIEs were calculated following:Northrop, D. B. In Enzyme mechanism from isotope effects; Cook, P. F., Ed.; CRC Press, 1991, pp 181-202.

39. Tunneling & Dynamics in theoretical modelsMarcus-like model of ground-state tunneling

40. Overview • Background and experimental tools • Dihydrofolate Reductase (DHFR) • Dynamics-activity relationship • Thymidylate Synthase (TS) • Alternative TS (FDTS)

41. Dihydrofolate Reductase

42. Thymidylate Synthase

43. Synthesis of Labeled Substrates

44. TS: Competitive Kinetic Assay H/T and D/T KIEs were measured. Intrinsic KIEs were calculated and their temperature dependence determined at 5-45 ˚C range. Nitish Agrawal, Cornelia Mihai, and Amnon Kohen*, Anal. Biochem.328, 44-50 (2004). Agrawal, N., Hong, B., Mihai, C., and Kohen, A.*Biochemistry, 43, 1998-2006 (2004).

45. TS: Arrhenius Plot of V/K KIEs: Observed H/T D/T

46. Arrhenius Plot of V/K KIEs: Observed vs. Intrinsic H/T D/T

47. Arrhenius Plot of V/K KIEs: Intrinsic H/T D/T

48. Arrhenius Plot of V/K KIEs: Intrinsic H/T D/T

49. Semiclassically Calculated Range for the KIE on Arrhenius Preexponential Factors AH/AT and AD/AT Schneider & Stern (1972) J.A.C.S., 94, 1517-1522. Stern & Weston, (1974) J.Chem. Phys.., 60, 2815-2821. Bell (1980) The Tunneling Effect in Chemistry, Chapman & Hall, ED., London & New York. Melander & Saunders (1987) Reactions Rates of Isotopic Molecules, Krieger, Ed., Fl.

50. TS : Temperature dependence of steady-state initial velocity data 40°C 30°C 20°C 5°C Values of the kcat were determined by fitting steady-state initial velocity data to the following substrate inhibition equation: V = kcat[S]/(Km + [S]* (1+[S]2/KS))