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Department of Chemistry The University of Iowa

Protein dynamics and tunneling effects in the DHFR and TS (ThyA) catalysis N. Agrawal, B. Hong, C. Mihai, L. Wang, S. Tharp, K.A. Markham, S.J. Benkovic and A. Kohen. Department of Chemistry The University of Iowa. Overview. General questions and experimental tools

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Department of Chemistry The University of Iowa

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  1. Protein dynamics and tunneling effects in the DHFR and TS (ThyA) catalysis N. Agrawal, B. Hong, C. Mihai, L. Wang, S. Tharp, K.A. Markham, S.J. Benkovic and A. Kohen Department of Chemistry The University of Iowa

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

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

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

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

  6. Size H [product] D time Temperature dependency AH/AT AD/AT 1.6 1.2 0.6 0.9 KIEs as Probe of Tunneling

  7. AH/AT AD/AT 1.6 1.2 0.6 0.9 KIE Arrhenius Plots Kohen, A.,Klinman, J. P.;(1999) Chemistry & Biology, 67, R191-R198

  8. E R.C. Kinetic complexity

  9. Thymine biosynthesis

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

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

  12. 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

  13. 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

  14. 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

  15. 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.

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

  17. 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

  18. Coupled 1˚-2˚ motion From the mixed labeling experiment Ln(1.19)/ln(1.052)=3.4 ±1 —No coupled motion Kohen, A. and Jensen J. J. Am. Chem. Soc. (2002) 124 3858-3864. Calculated vs. experimental 2˚ H/D KIEs Calculated : 1.13 Experimental: 1.13 ± 0.02 Equilibrium: 1.127 ± 0.009 Location of the transition state?

  19. 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.

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

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

  22. Dihydrofolate Reductase

  23. Thymidylate Synthase

  24. Synthesis of Labeled Substrates

  25. TS: Competitive Kinetic Assay

  26. KIE Determination KIEs (L/T) were calculated from experimental T/14C ratios (Rt and R∞)and fractional conversions (f): Nitish Agrawal, Cornelia Mihai, and Amnon Kohen*, Anal. Biochem. (2004), In Press The intrinsic KIE was calculated by Northrop’s method and their temperature dependence determined at 5-45 ˚C range.

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

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

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

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

  31. 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.

  32. 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))

  33. TS : Temperature dependence of steady-state initial velocity data 40°C 30°C 20°C 5°C 1.8 Ea = 4 ± 0.1 Kcal/mol 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))

  34. AH/AT AD/AT 1.6 1.2 0.6 0.9 KIE Arrhenius Plots Agrawal, N., Hong, B., Mihai, C., and Kohen, A.*Biochemistry, 43, 1998-2006 (2004).

  35. Vibrationally Enhanced Tunneling

  36. Vibrational wave functions of the transferring hydride for representative configurations. On the donor side, the donor carbon atom and its first neighbors are shown, whereas on the acceptor side, the acceptor carbon atom and its first neighbors are shown. The ground and excited vibrational states are shown on the left and right, respectively. Hammas-Shiffer and co-workers J. Phys.Chem. B (2002) 106, 8283-8293.

  37. 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.

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

  39. DHFR Temperature Dependency - w.t. vs. G121V Commitment 2.8±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.

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

  41. Relevant Protein Motion

  42. Results and Conclusions • DHFR: • 1. The lack of temperature dependence of intrinsic primary KIEs constitutes proof for hydrogen tunneling, and taken together with the Ea suggests vibrationally enhanced H-tunneling. • 2. The secondary intrinsic KIEs were 1.19 ±0.015 and 1.052 ±0.019 for H/T and D/T, respectively, which doesn’t suggest 1˚-2˚ coupled motion (the Swain-Schaad EXP = 3.4 ±1). • 3. The intrinsic KIEs at 25 ˚C are in agreement with the values predicted by Truhlar, Gao, Hammes-Schiffer and their co-workers from QM/MM calculations. • 4. The dynamically altered mutant G121V catalyze similar H-transfer mechanism but its pre-organization is not perfect and some “gating” is required. • * Sikorski, et al., J. Am. Chem. Soc., 126, 4778-4779 (2004). • TS: • 1. The lack of temperature dependence of intrinsic primary KIEs constitutes proof for hydrogen tunneling, and taken together with the Ea suggests vibrationally enhanced H-tunneling. • 2. Substrate inhibition was predicted but is observed here for the first time. This demonstrates that relevant activation parameters can only be calculated on a single rate constant (e.g., kcat) if it is extracted from the whole kinetic cascade at all temperatures. • * Agrawal, et al., . Biochemistry, 43, 1998-2006 (2004).

  43. Summary and Future Directions • Methods were developed to examine the nature of the hydride transfer step in complex enzymatic reaction cascades, which allow measurements of 1º and 2º, H/T and D/T KIEs on V/K, their commitments to catalysis and of intrinsic KIEs. • These methods expose the nature of the chemical step better than previous experiments. • Both TS and DHFR catalyzed reactions seem to involve “environmentally coupled tunneling” but no coupled motion between 1˚ and 2˚ hydrogens. • Current and future studies: • Various DHFR mutants with altered dynamics are studied to examine possible effects of those dynamics on the nature of the hydride transfer (e.g., G121V, M42W, and G121V-M42W). • Alternative DHFRs and TSs, such as R67-DHFR, TSCP or thermophiles and halophiles, will be studied and the nature of their chemical transformations will be compared. The relationship between sequence, structure, dynamics and function (H-transfer catalysis) will be examined.

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