Introduction. The dynamic nature of enzyme function can be viewed as extended induced fit, which includes

Transcription

1 Introduction While the last century brought us to a closer understanding of enzyme catalysis, the traditional lock-and-key model has been gradually replaced by the views of a more flexible active site. The dynamic nature of enzyme function can be viewed as extended induced fit, which includes not only the conformational changes upon substrates binding, but also fast protein motions along the reaction coordinate of the chemical transformations (chemical steps). 1-3 Although immense experimental and theoretical effort has been devoted to this field, the relationship between protein motions and enzyme catalysis still remains to be an unsolved question. Understanding this relationship is important because enzymes are targets of numerous antibiotic and chemotherapeutic drugs, as well as models for artificial catalysts designs. The difficulty in resolving the motions-catalysis relationship lies in the fact that the transition state (TS) of an enzyme-catalyzed chemical reaction is very unstable (i.e. disappears within a few femtoseconds) and cannot be directly observed. Furthermore, many enzyme-catalyzed chemical steps are not the rate-limiting step of the overall catalysis, and therefore other slower steps usually mask the information on the chemical steps. Valuable insights to the motionscatalysis relationship have come from recent studies on hydrogen transfers in various enzymatic systems, using temperature dependence of the kinetic isotope effect (KIE). 4-7 KIE can measure the kinetic properties directly relevant to the TS of a specific chemical step without being masked by other steps. Since an elevation in temperature will affect some protein motions, one can investigate the role of enzyme motions during catalysis by measuring the KIEs in a physiological temperature range (5 40 o C). Previous studies have reported temperatureindependent KIEs in various wild-type enzymes with their natural substrates under physiological conditions However, when the enzymes work under other conditions (e.g. by mutation, 12 non- 1

2 physiological temperatures 10 ), they exhibit temperature-dependent KIEs. Several theoretical calculations and simulations have examined the temperature dependence of KIEs in these enzymatic systems Although the details of the calculations and simulations are diverse, they agree on the basic notion that the temperature dependence of KIEs reflects the temperature dependence of the donor-acceptor distance (DAD) at the TS for the hydrogen transfer. More explicitly, the wild type enzymes seem to employ different types of motions to reach an optimal TS for hydrogen to transfer efficiently, and this transition state is characterized by a narrow distribution of DAD that does not fluctuate with temperature. A recent study on formate dehydrogenase with both KIE and time-resolved photon-echo experiments corroborates this suggestion. 9,16 In this work we study thymidylate synthase (TSase, EC ), which catalyzes the reductive methylation of 2 -deoxyuridine-5 -monophosphate (dump) to form 2 - deoxythymidine-5 -monophosphate (dtmp, one of the four DNA bases) using N 5,N 10 - methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 folate) as both the methylene and hydride donor (Scheme 1). 17 Since TSase is essential for the de novo synthesis of DNA in nearly all organisms including humans, it is a common target for many antibiotic and chemotherapeutic drugs (e.g. Tomudex and 5-fluorouracil). The mechanism of TSase comprises a series of bond cleavages and formations including two different C-H bond activations (Scheme 1): a non-rate-limiting proton transfer (step 4) and a rate-limiting hydride transfer 8,18 (step 5). In order to understand the mechanism of TSase and to investigate the potential roles of protein motions in different hydrogen transfer steps, we have performed both KIE experiments and quantum mechanics/molecular mechanics (QM/MM) calculations on the two hydrogen transfers catalyzed by TSase. 2

3 Methodology [2-14 C] dump (specific radioactivity 56 Ci/mol) and [5-3 H]-dUMP (specific radioactivity 13.6 Ci/mmol) were from Moravek Biochemicals. Ultima Gold liquid scintillation cocktail were from Packard Bioscience. Liquid scintillation vials were from Fisher Scientific. The wild-type E. coli thymidylate synthase (TSase) was expressed and purified following the published procedure. 19 We used an Agilent Technologies model 1100 HPLC system for all the purifications and analytical separations. All other materials were purchased from Sigma. Competitive KIE Experiments In order to better expose the KIEs of the non-rate-limiting proton transfer step, we modified the experiment conditions based on the previously developed method. 20 The experiments were performed in 100 mm tris(hydroxymethyl)aminomethane (Tris)/HCl buffer (ph 7.5, adjusted at the desired temperature), at 5, 15, 25, and 35 C. Each reaction mixture (final volume 1 ml) contained 50 mm MgCl 2, 1 mm EDTA, 5 mm formaldehyde, 25 mm DTT, 3 μm CH 2 H 4 folate, 0.9 Mdpm [5-3 H] dump, and 0.3 Mdpm [2-14 C] dump (for H/T KIE measurement) or [2-14 C, 5-2 H] dump (for D/T KIE measurement). At each temperature, the experiments were performed following the previously published procedure. 20 Two independent control experiments (minus enzymes) were performed to deduct the non-enzymatic effects. All the quenched samples were analyzed by RP HPLC separation and liquid scintillation counting (LSC) to obtain the observed KIEs, as described in previous publications. 8 All t time points were pooled together to provide more accurate average values. The competitive observed KIEs were determined from three measured values the fraction conversion (f), the 3 H/ 14 C ratio in the products ([ 3 H]H 2 O and [ 14 C]dTMP) at each time point (R t ), and the 3 H/ 14 C ratio in the products at the infinity time point (R ): 4,21 3

4 ( ) ln 1" f KIE = % ln 1" f # R ( t ' * & ) R $ (6) The fraction conversion f was calculated by: f = [ 14 C]dTMP [ 14 C]dTMP +[ 14 C]dUMP (7) The intrinsic KIEs were calculated using the Northrop method: 4,22 T T ( V /K) "1 Hobs "1 V /K "1 "1 = k T k H "1 ( ) Dobs ( k T k H ) 1/ 3.34 "1 (8) where T ( V /K) Hobs and T ( V /K) Dobs are the observed H/T and D/T KIEs, respectively; k T k H represents the reciprocal of the intrinsic H/T KIE. The intrinsic H/T KIE was solved numerically. All possible combinations of observed H/T and D/T KIEs were analyzed to get the intrinsic KIEs. All intrinsic KIEs were used to fit to the Arrhenius equation (Eq. 5) to evaluate the isotope effects on Arrhenius parameters and temperature dependence of KIEs. This analysis was carried out with KaleidaGraph (Version 4.03) as the least root-mean-square fit exponential regression. QM/MM calculations The starting protein structure was obtained from the PDB entry 1TLS, which is a ternary complex of E. coli TSase with the substrate dump and the cofactor CH 2 H 4 folate. The pk a value of ionisable amino acids was calculated using the program PropKa, and the protonation states were determined at ph of 7.5 (same with the experimental conditions). A total of 24 sodium counterions were placed in optimal electrostatic positions around the enzyme because the total charge of the system was not neutral. Finally, the system was solvated using a box of water molecules of Å 3 (Figure 2). The whole system was divided into a QM part and a MM part to perform the QM/MM calculations (Scheme 2). 23 The QM atoms of the system are described by the semiempirical Austin Model 1 (AM1) 24 Hamiltonian, 4

5 while the atoms of the MM region by the opls and TIP3P 25 force fields. A few calculations were repeated with the QM part described by a density functional theory (DFT)-based method with a BLYP exchange functional and a 6-31(+)G** basis set, to verify the reliability of the AM1 calculation results. Results and Discussions Intrinsic KIEs on the Non-rate-limiting Proton Transfer One of the main challenges in enzyme studies is to expose and examine a chemical step that is not rate limiting within the complex catalytic cascade. While the hydride transfer is rate-limiting for both the first and second order rate constants (k cat and k cat /K M, respectively) for dump, 8 the proton transfer is a reversible nonrate-limiting step and is more difficult to study. A previous study monitored the release of [ 3 H]H 2 O and production of [ 14 C]dTMP using [2-14 C, 5-3 H]dUMP as the substrate with a saturating concentration of CH 2 H 4 folate, which reported a KIE of unity (i.e. no isotope effect) on the proton transfer. 26 We have suggested that the observed KIEs for the fast proton transfer are dependent on the concentration of CH 2 H 4 folate, due to the sequential binding of dump and CH 2 H 4 folate to the enzyme (Scheme 2). 20 Thus, an accurate assessment of the KIEs on the proton transfer requires optimal reaction conditions in which the concentration of CH 2 H 4 folate is high enough to ensure sufficient conversion of dump to dtmp while low enough to ensure that the observed KIEs will not be masked by the forward commitment to catalysis (see below). 4,22 We improved the competitive KIE methods previously developed in our lab (see Experimental Section), and measured observed H/T and D/T KIEs on the proton transfer. Figure 1A shows the observed and intrinsic KIEs on the proton transfer 27, and 1B the hydride transfer 8. The intrinsic KIEs were calculated from the observed KIEs using the Northrop method (see Methodology). In 5

6 contrast with the hydride transfer, the observed KIEs on the proton transfer are much smaller than the intrinsic KIEs, suggesting other slower steps mask the intrinsic KIEs on this step. 4,22 Comparison of the Temperature Dependences of Intrinsic KIEs on the Proton and Hydride Transfers The intrinsic KIEs on both the proton and hydride transfers were fit to the Arrhenius equation to evaluate their temperature dependences: 28 KIE = k L = A $ L exp " #E a & k H A H % RT ' ) (5) ( where the subscripts L and H denote a light and a heavy isotope of hydrogen, respectively; k represents the microscopic rate constant of the isotopically sensitive step (step 4 for the proton transfer and step 5 for the hydride transfer in Scheme 1, respectively), and ΔE a is the difference in the activation energies of that microscopic step between the light and heavy isotopes; R is the gas constant and T is the absolute temperature. Therefore, the isotope effects on both the preexponential factors (A L /A H ) and the activation energy (ΔE a ) can be accessed by fitting the intrinsic KIEs to Eq. 5 (Figure 1). The isotope effects on the Arrhenius parameters of both the proton and hydride transfers are summarized in Table 1. The intrinsic KIEs on the hydride transfer are temperature-independent, and the isotope effects on the pre-exponential factors are well above the semi-classical limits. 4 In contrast, the KIEs on the proton transfer are temperature-dependent and A L /A H s are much lower than the semi-classical limit. QM/MM calculations on the mechanisms of the proton transfer and the following steps Since the proton transfer is a fast step, there have been very limited experimental data regarding the mechanism of this step. In the traditionally proposed mechanism, the proton abstraction either leads to an enolate formation at the C4 position of dump (Scheme 2 Blue). We have examined several different mechanisms for the proton transfer step with QM/MM calculations, and the results suggest this step is more likely to occur through transient cleavage of the C6-S bond 6

7 between dump and Cys146, which forms an intermediate with C5-C6 double bond (Scheme 2 Red). More interestingly, the cleavage of the C6-S bond seem to be concerted with other steps following the proton transfer and serves as a mechanistic feature throughout the reaction. The C6-S bond is the only covalent bond between the substrate and the protein during TSase catalysis, and has been the primary basis for drug designs (e.g. 5-flurine-dUMP). The new proposed mechanism suggests several intermediates that have not been proposed before (Scheme 2 Red), which can provide novel directions for drug designs that target TSase. Insignts from QM/MM calculations on the temperature dependence of KIEs Our previous calculations 29 have reproduced temperature-independent KIEs and revealed the average DAD for the hydride transfer does not fluctuate with temperature (Table 1), which corroborates with the previous suggestions that the temperature dependence of KIEs reflect the temperature dependence of DADs at TS. The current work revealed a water molecule is the acceptor for the proton transfer step (Figure 3). As water molecules are more mobile than the protein residues and the bound substrate/cofactor, it is expected that the DADs at TS for the proton transfer will fluctuate with temperature, which can explain the temperature-dependent KIEs for the proton transfer step. Protein motions revealed in QM/MM calculations Our QM/MM calculations have found the cleavage of the C6-S bond between the substrate and the protein is concerted with a sequence of chemical steps during TSase catalysis. The lability of this C6-S bond is assisted by the nearby Arg166 residue, which polarizes the Cys146 residue. Figure 3 shows the representative snapshots from the reactant, transition state, and product for the proton transfer, to demonstrate the cleavage of C6-S bond during the concerted chemical steps, and the relative movements of Arg166 and Cys146. Similar protein motions were also found for the hydride transfer step. 30 7

8 Conclusions and future directions We analyzed and compared the nature of two sequential H-transfers catalyzed by TSase a non-rate-limiting proton transfer and a rate-limiting hydride transfer. The temperature dependences of intrinsic KIEs on the two H-transfers are dramatically different, which suggests different activation mechanisms. Based on the QM/MM calculations, the DAD at the TS for the hydride transfer step does not fluctuate with temperature, which corroborates with the previous suggestions that the temperature dependence of KIEs reflect the temperature dependence of DADs at TS. An active site water molecule is the acceptor for the proton transfer, which suggests the DADs at TS will fluctuate with temperature. Further calculations are in progress to calculate the temperature dependence of both DADs and KIEs for the proton transfer step. Our calculations also revealed a new mechanism for the proton transfer step, which may lead to a new direction for drug designs that target TSase. Arg166 is critical in the new mechanism but not in the traditional mechanism, therefore, we are also studying the kinetics of its mutants (Arg166Lys and Arg166Ala) to verify the proposed mechanism. Furthermore, our calculations revealed protein motions that are concerted with a sequence of chemical steps during catalysis, and provide insights into the motions-catalysis relationship in enzymatic systems. 8

9 Tables, Schemes, and Figures Table 1. The average DADs at the TS for the hydride transfer step do not change with temperature (5-40 C). T (K) average DADs (Å) ± ± ± ±

10 10

11 References (1) Benkovic, S. J.; Hammes-Schiffer, S. Science 2003, 301, (2) Boehr, D. D.; McElheny, D.; Dyson, H. J.; Wright, P. E. Science 2006, 313, (3) Henzler-Wildman, K.; Kern, D. Nature 2007, 450, 964. (4) Kohen, A. In Hydrogen-Transfer Reactions; Hynes, J. T., Klinman, J. P., Limbach, H.-H., Schowen, R. L., Eds.; Wiley_VCH: Weinheim, 2007; Vol. 4, p (5) Nagel, Z. D.; Klinman, J. P. Nat Chem Biol 2009, 5, 543. (6) Schwartz, S. D. In Isotope effects in chemistry and biology; Kohen, A., Limbach, H. H., Eds.; Taylor & Francis, CRC Press: Boca Raton, FL, 2006; Vol. Ch. 18, p 475. (7) Truhlar, D. G. In Isotope effects in chemistry and biology; Kohen, A., Limbach, H. H., Eds.; Taylor & Francis, CRC Press: Boca Raton, FL, 2006; Vol. Ch. 22, p 579. (8) Agrawal, N.; Hong, B.; Mihai, C.; Kohen, A. Biochemistry 2004, 43, (9) Bandaria, J. N.; Cheatum, C. M.; Kohen, A. J Am Chem Soc 2009, 131, (10) Kohen, A.; Cannio, R.; Bartolucci, S.; Klinman, J. P. Nature 1999, 399, 496. (11) Sikorski, R. S.; Wang, L.; Markham, K. A.; Rajagopalan, P. T.; Benkovic, S. J.; Kohen, A. J Am Chem Soc 2004, 126, (12) Wang, L.; Goodey, N. M.; Benkovic, S. J.; Kohen, A. Proc. Natl. Acad. Sci. USA 2006, 103, (13) Edwards, S. J.; Soudackov, A. V.; Hammes-Schiffer, S. The Journal of Physical Chemistry A 2009, 113, (14) Liu, H.; Warshel, A. J Phys Chem B 2007, 111, (15) Meyer, M. P.; Klinman, J. P. Chemical Physics 2005, 319, 283. (16) Bandaria, J. N.; Dutta, S.; Hill, S. E.; Kohen, A.; Cheatum, C. M. J Am Chem Soc 2008, 130, 22. (17) Carreras, C. W.; Santi, D. V. Annu. Rev. Biochem. 1995, 64, 721. (18) Spencer, H. T.; Villafranca, J. E.; Appleman, J. R. Biochemistry 1997, 36, (19) Changchien, L.-M.; Garibian, A.; Frasca, V.; Lobo, A.; Maley, G. F.; Maley, F. Prot. Expres. Pur. 2000, 19, 265. (20) Hong, B.; Maley, F.; Kohen, A. Biochemistry 2007, 46, (21) Melander, L.; Saunders, W. H. Reaction rates of isotopic molecules; 4th ed.; Krieger, R. E: Malabar, FL, (22) Northrop, D. B. In Enzyme mechanism from isotope effects; Cook, P. F., Ed.; CRC Press: Boca Raton, FL., 1991, p 181. (23) Warshel, A.; Levitt, M. Journal of Molecular Biology 1976, 103, 227. (24) Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. Journal of the American Chemical Society 1985, 107, (25) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Journal of Chemical Physics 1983, 79, 926. (26) Santi, D. V.; Stroud, R. M. Biochemistry 2002, 42, 248. (27) Wang, Z.; Kohen, A. J. Am. Chem. Soc. 2010, 132, (28) Stroud, R. M.; Finer-Moore, J. S. Biochemistry 2003, 42, 239. (29) Kanaan, N.; Ferrer, S.; MartiÃÅ, S.; Garcia-Viloca, M.; Kohen, A.; Moliner, V. J. Am. Chem. Soc. 2011, 133, (30) Kanaan, N.; Ferrer, S.; Marti, S.; Garcia-Viloca, M.; Kohen, A.; Moliner, V. J. Am. Chem. Soc. 2011, 133,