J. Phys. Chem. 1995, 99, 6273-6276
6273
Ribonuclease A Catalyzed Transphosphorylation: An ab Znitio Theoretical Study Brian D. Wladkowski,**+Morris Krauss, and Walter J. Stevens Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland 20850
Received: February 20, I995@
The ribonuclease A transphosphorylation mechanism is studied using a b initio quantum chemical methods, incorporating for the first time detailed all-electron components which mimic important amino acid residues within the enzyme active site. The 2-hydroxyethyl methylphosphate monoanion is chosen as a model substrate, methyleneimines (CHzNH) are used in place of the imidazole rings for His-12 and His-119, and methylamine (CH3NH2) is used in place of Lys-41. Each pseudoresidue is held fixed in its appropriate relative position found crystallographically. Within this model, structures and relative energetics for the stationary points along the transphosphorylation reaction pathway are determined at the RHF level using a 3-21G+* basis set. The data reveal several low-barrier proton transfer steps between the substrate and the active site residues which allow transphosphorylation to occur with modest activation, consistent with experimental results for the actual enzyme. Two distinct aspects of the active site are identified: a general acid to help protonate the substrate and an acidhase pair which cooperatively facilitates proton transfer as transphosphorylation takes place.
Introduction
step in the RNase A mechanism. Although their work has provided valuable insight into phosphate reactivity, its applicability to the RNase A mechanism is somewhat limited without explicit consideration of critical components from the enzyme active site in the chemical model. We believe such considerations are essential for the application of ab initio quantum techniques to study enzyme-catalyzed reaction mechanisms. In this Letter, we report ab initio quantum chemical results on a model transphosphorylation reaction, incorporating for the first time detailed all-electron components which mimic important amino acid residues within the RNase A active site. The results presented here help to identify the specific microscopic roles important active site residues play in the RNase A enzymecatalyzed mechanism.
Pancreatic ribonuclease A (RNase A), an efficient enzyme catalyst for the hydrolysis of phosphate-ester linkages in singlestranded RNA, has been the subject of extensive experimental’ and t h e ~ r e t i c a l ~investigation -~ for more than 50 years. The catalytic process is known to occur via a two-step general-acid base mechanism. The first step involves intramolecular transphosphorylation leading to a cyclic phosphate intermediate and displacement of the 05’-nucleotide product. In the subsequent step, the cyclic phosphate is hydrolyzed to a 3‘-monophosphate nucleotide as the final product. Two amino acid residues within the active site of RNase A, His-12 and His-119, are known to play critical roles in the catalytic machinery and are believed to act as the general base and general acid, respectively, in the f i s t step of the reaction, reversing their roles in the second step. Despite the wealth of available experimental data, however, Quantum Chemical Model and Computational Details RNase A continues to attract considerable attention. The specific role of Lys-41, an active site residue necessary for full The chemical model used here to probe the RNase Acatalytic activity, and details concerning the individual proton catalyzed transphosphorylation mechanism consists of a simple transfer steps, as well as the protonation state within the active phosphate-ester substrate, 2-hydroxyethyl methylphosphate site at each stage of the reaction, are all issues of much debate. monoanion (HEMP), capable of undergoing intramolecular The general uncertainty surrounding the details of the RNase transphosphorylation to ethylene phosphate (EP) plus methanol, A catalytic mechanism is further highlighted by the recent as well as essential parts of the three important amino acid experimental work of Breslow and co-worker@which suggests residues found in the RNase A active site (Scheme 1). HEMP the true microscopic mechanism may be more complex than represents the smallest subunit of a complete dinucleotide with previously thought. Recent experimentalefforts to resolve these the necessary components required to undergo transphosphoas well as other issues related to the RNase A catalytic rylation and is the same substrate as used by Lim and Tole4fin mechanism have been unsuccessful.7~8 their initial study, which allows for a direct comparison with In an attempt to generate a more coherent and detailed picture the earlier work. The three important active site residues, Hisof how RNase A functions microscopically, many researchers 12, His-119, and Lys-41, are represented by small molecular have applied various computationaltechniques over the year^.^-^ subunits chosen to mimic the important features of each. HisInitial work relied primarily on molecular mechanic^^^^^^ and 12 and His-119 are represented by methyleneimines (His-12, semiempiricalmethods;zb~c however, in more recent s t u d i e s ? ~ ~ ~ - ~ His-119 CHzNH), and Lys-41 is represented by a methylab initio quantum chemical methods have been applied to amine (Lys-41 CH3NH2). Incorporation of such “pseudoisolated model substrates as a way to probe the actual enzyme residues” directly into the chemical model allows for any mechanism. Lim and Tole4f were one of the first to use ab necessary electron rearrangement, including proton transfer, initio methods in this way to study the transphosphorylation between the active site components and the substrate, while maintaining a practical sized system. The complete chemical + National Research Council (NRC) Postdoctoral Fellow. system is assumed to have an overall plus one charge, with a @Abstractpublished in Advance ACS Abstracts, April 15, 1995.
- -
This article not subject to U.S. Copyright. Published 1995 by the American Chemical Society
Letters
6274 J. Phys. Chem., Vol. 99, No. 17, 1995 SCHEME 1 H
H I
His-119
I
I
His-12
H
His-12 H3C
Lys-41
monoanionic substrate, two positively charged active site residues, and one neutral residue, consistent with expectations based on estimated pKa data for the enzyme prior to substrate binding. The constraints used to fix the relative position of components in the chemical system are another important consideration for the construction of a physically meaningful model. Here, coordinates from a recent high-resolution X-ray crystallographic study of the RNase Nuridine vanidate (UV) complex9 are used to determine the appropriate relative constraints for each pseudo-residue in the chemical model. It is important to recognize, however, that no internal constraints are imposed on any species, and although the relative orientation of each pseudo-residue is maintained, the substrate is unconstrained. Using the complete chemical system described above, optimized structures and relative energetics for salient stationary points along transphosphorylation reaction pathway were determined. The reaction pathway was explored starting from the most stable pentacoordinate trigonal-bipyramid (TBP) phosphorane structure (shown in Figure 1) by elongation of the endocyclic 02'-P bond toward reactant or exocyclic 05'-P bond toward product. The most stable structure at each stage of the reaction was then determined by movement of the protons to and from the substrate. Results were obtained at the restricted Hartree-Fock (RHF) level using the 3-21+G* basis set of Pople and co-workers.1° All calculations were performed using the Gaussian 9211 suite of electronic structure codes. For the chemical system described above, the 3-21+G* basis set consists of 239 contracted Gaussian functions, and each stationary point was located using analytic gradients and verified as a true minimum or transition state via analytic second-derivative calculations.
Figure 1. Diprotonated phosphorane intermediate structure, I4*, optimized at the RHF 3-21+G* level, showing hydrogen bond interactions and atom labeling.
I
His-119
0 H3C
Lys-41
Results and Discussion The RHF optimized structures and related connectivity for each local minimum along the transphosphorylation reaction pathway for the chemical system in Scheme 1 are given in Figure 2. More detailed structural information for each stationary point is given in Table 1, and relative energetic data (AEeo, kcal mol-') at the RHF level are shown schematically in Figure 3. As expected, the qualitative reaction coordinate for transphosphorylation involves contraction of the 02'-P bond and an elongation of the 05'-P bond from approximately 3.4 and 1.6 A, respectively, in the reactants to approximately 1.7 and 1.9 A,respectively, in the product complex (see Table 1). As shown in Figure 2, however, the overall reaction pathway is quite complex containing a number of discrete intermediate states involving proton transfer between the His-12, His-1 19, and Lys41 pseudo-residues and the substrate. Interestingly, the most stable intermediate along the transphosphorylation pathway within the model used here is the diprotonated (neutral) TBP phosphorane structure, I4*, and not the monoanionic or dianionic phosphorane as has been proposed previously. However, 14" does not lead directly to reactants or products. The most relevant TBP structure to the transphosphorylation pathway is the monoanionic phosphorane, 13, in which a proton has been transferred .from the substrate back to N2 on Lys-41. From intermediate 13, the two pathways energetically accessible are exocyclic displacement toward products and endocyclic displacement back toward reactants. The exocyclic pathway through structure EXTS involves proton transfer between the His-1 19 residue and 05' of the substrate with concerted breakage of the 05'-P bond, consistent with previous proposals for the mechanism. EXTS leads directly to the product complex P, representing the association complex between methanol and protonated ethylene phosphate (EPH). Alternatively, the endocyclic channel from intermediate I3 via elongation of the 02'-P bond from 1.9 to 2.7 A through the transition state structure ENTS leads to the intermediate structure 12. Structure I2 can be formally considered an alkoxy phosphate anion in which charge has been transferred from the phosphoryl oxygen 0 1 to 02'. From the alkoxy structure 12, two energetically comparable pathways are possible, both of which lead to the reactant, HEMP. One pathway involves intramolecular proton transfer between the phosphoryl oxygen 0 2 and the alkoxyoxygen 02', through PTTS3, leading directly to the reactant complex, R2. In the transition state structure PTTS3, His-1 12 helps to facilitate the proton transfer but does not actually participate directly. An alternative pathway from intermediate I2 involves initial proton transfer from the Lys-41 catalytic nitrogen N2 to the alkoxy oxygen 02' on the substrate through the transition state structure PTTS2, to form intermediate 11, followed by proton transfer from the phosphoryl oxygen 0 2 on the substrate back to His-12 through structure PTTS1,
Letters
J. Phys. Chem., Vol. 99, No. 17, 1995 6275
14*
I3
R1
P
I1
Figure 2. Important minimum-energy structures along the transphosphorylation reaction pathway determined at the RHF 3-21+G* level (see text). TABLE 1: Important Geometric Parameters (A) for the Stationary Points along the Transphosphorylation Reaction Pathway at the RHF 3-21+G* Level stationary point r(P-02’) r(P-01) r(P-02) r(P-03’) r(P-05’) r(N1-02’) r(N2-02’) r(N1-02) r(N2-01) r(N3-05’) R1 3.4529 1.4771 1.5163 1.6159 1.6282 3.66 2.64 3.32 2.71 2.83 R2 3.3564 1.4741 1.5212 1.6231 1.6150 4.732 3.227 3.427 2.702 4.076 I1 3.5033 1.4658 1.5558 1.5867 3.826 2.854 2.682 3.266 2.763 1.6033 I2 2.661 1 1.4792 1S606 1.5855 3.1 17 2.842 3.322 2.7 16 2.700 1.6242 I3 1.8694 1.5246 1.5896 1.6393 1.6646 2.648 3.235 2.897 2.642 3.195 14* 1.7664 1.5652 1.5836 1.6283 3.170 1.6537 3.113 2.6 19 2.727 3.002 PTTSl 3.4403 1.4692 1.5442 1.5983 1.6112 3.262 2.739 3.763 2.844 2.544 PTTS2 2.6782 1.4724 1.5610 1S876 1.6230 3.23 1 2.764 3.330 2.679 2.760 F‘TTS3 2.9680 1.4379 1.5431 1S849 3.637 2.827 1.6086 3.252 2.742 3.035 PTTS4 1.8299 1.5380 1.5879 1.6417 1.6566 3.163 2.932 2.63 1 2.499 3.430 ENTS 2.2639 1.4921 1.5655 1.5997 1.6497 3.254 2.812 2.652 2.828 3.062 EXTS 1.8830 1.4922 1.5887 1.6159 3.148 2.558 1.7621 3.188 2.903 2.653 P 1.7285 1.5051 1S685 1.6090 1.8925 3.176 2.6 15 2.664 3.212 2.886 PITS3
0.0
1.1
Figure 3. Relative energy diagram (kcal mol-’) for the stationary points along the transphosphorylation reaction pathway determined at the RHF 3-2 1+G* level.
6276 J. Phys. Chem., Vol. 99, No. 17, 1995 leading to reactant structure R1. As can be seen from Figure 1, the only difference between reactant complexes R1 and R2 is the position of the proton. In structure R1 the proton starts out on His-12, whereas in structure R2 it starts out on Lys-41. Considering the relative energetic data in Figure 3, several important results become immediately apparent. (1) The overall activation energy calculated at the RHF 3-21+G* level for this model transphosphorylation reaction is less than 20 kcal mol-' and considerably lower than estimates based on the model substrate alone.4f Moreover, the final energetic results are qualitatively consistent with experimental kinetic data for the actual RNase A enzymatic transphosphorylation step (kcat = 1-3000 s-l)l for simple dinucleotide substrates. (2) There exists a number of intermediate states only slightly higher in energy than the reactant complex including the TBP phosphorane structures. The diprotonated phosphorane structure 14* and the alkoxy intermediate I2 are only 5 and 8 kcal mol-', respectively, higher in energy than the reactant complex R1. In the case of the intermediate state I4*,its dramatic stability results from an increase in the proton affinity of the pentacoordinate phosphorane anion compared to that of the reactant phosphate anion.12 As a result, the phosphorane can be stabilized as long as active site residues (either Lys-41 or His-12) are available and positioned appropriately. (3) The initial reactant states in which the proton resides on either Lys-41 or His-12 are within 3 kcal mol-', yet the most stable structure, R1, has the proton on His-12 and not Lys-41, at variance with pKa expectation^.'^ Moreover, each initial reactant state can lead to product formation through different channels of comparable energy. This suggests that Lys-41 can play an intimate role in the catalytic process (Le., general base) as opposed to simply stabilizing the intermediate phosphorane as has been proposed. In fact, the reaction channel starting from R1 is the overall lowest energy channel within this simplified m0de1.l~ (4) Finally, an important feature of transphosphorylation identified in both reaction channels is initial protonation of a nonbridging phosphoryl oxygen on the substrate prior to nucleophilic attack at phosphorus. This is consistent with, but slightly different from, the mechanism proposed by Breslow, in which His-1 19 is the initial protonating species. In conclusion, we have identified two critical features of the RNase A active site environment (a general acid to neutralize the substrate and a general acidgeneral base pair to facilitate proton transfer) which promote transphosphorylation reactions with modest activation energy. In the presence of a sufficient environment that allows for a specific reaction pathway, the intrinsic barrier to transphosphorylation for the model system used here is found to be comparable to the RNase A enzymecatalyzed kinetic results. Viewed in this way, the function of RNase A is more than simply to mediate the corresponding gasphase or solution-phase reaction through nonbonded interactions. The key residues can act to chemically participate through catalytic proton transfer, giving rise to a microscopic mechanism that may be otherwise inaccessible. The concept of intimate interaction between active site residues within RNase A and the reacting system is certainly not new. Gerlt et al.15 have argued the importance of strong hydrogen bond formation between groups with similar pKa's as a way to help stabilize critical structures along the reaction pathway. Lim and Tole4f have also postulated intimate interactions between the substrate and active site residues and have even speculated on specific
Letters roles for each residue in mediating the energetics of the anionic reaction they studied computationally. Finally, we recognize that in the case of RNase A, other residues besides His-12, His119, and Lys-41, or combinations of residues may satisfy the important environmental components identified here. Computational studies utilizing an even more complete model of the RNase A active site to probe the microscopic mechanism are currently underway. Acknowledgment. We are grateful to Jan Jensen and Professor Mark Gordon (Iowa State University) for helpful discussion. References and Notes (1) For reviews, see: (a) Richards, F. M.; Wyckoff, H. W. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1971; Vol. 4. (b) Blackbum, P.; Moore, S. The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1982; Vol. 15. (2) (a) Homes, R. R.; Deiters, J. A,; Gallucci, J. C. J. Am. Chem. SOC. 1978,100,7393. (b) Deakyne, C. A.; Allen, L. C. J. Am. Chem. SOC.1979, 101, 3951. (c) Umeyama, H.; Nakagawa, S.; Fujii, T. Chem. Pharm. Bull. 1979, 27, 974. (3) (a) Taira, K.; Uebayasi, M.; Maeda, H.; Furukawa, K. Protein Eng. 1990, 3, 691. (b) Uchimaru, T.; Tanabe, K.; Nishikawa, S.; Taira, K. J . Am. Chem. SOC.1991,113,4351. (c) Taira, K.; Uchimam, T.; Tanabe, K.; Uebayasi, M.; Nishikawa, S. Nucleic Acid Res. 1991, 10, 2747. (d) Storer, J. W.; Uchimaru, T.; Tanabe, K.; Uebayasi, M.; Nishikawa, S.; Taira, K. J . Am. Chem. SOC.1991,113,5216. (e) Yliniemela, A.; Uchimam, T.; Tanabe, K.; Taira, K. J . Am. Chem. SOC. 1993, 115, 3032. (4) (a) Haydock, K.; Lim, C.; Brunger, A. T.; Karplus, M. J . Am. Chem. SOC.1990, 112, 3826. (b) Lim, C.; Karplus, M. J . Am. Chem. SOC. 1990, 112, 5872. (c) Dejaegere, A,; Lim, C.; Karplus, M. J . Am. Chem. SOC. 1991, 113,4353. (d) Lim, C.; Tole, P. J . Phys. Chem. 1992, 96, 5217. (e) Lim, C.; Tole, P. J . Phys. Chem. 1992, 97, 6212. (f) Lim, C.; Tole, P. J . Am. Chem. SOC. 1992, 114, 7245. (g) Dejaegere, A.; Karplus, M. J . Am. Chem. SOC.1993, 115, 5316. (h) Dejaegere, A,; Liang, X.; Karplus, M. J . Chem. Soc., Faraday Trans. 1994, 90, 1. ( 5 ) Krauss, M.; Basch, H. J . Am. Chem. SOC. 1992, 114, 3630. (6) For a review, see: Breslow. R. Ace. Chem. Res. 1991, 24, 317. (7) Herschlag, D. J. Am. Chem. SOC. 1994, 116, 11631. (8) Thompson, J. E.; Raines, R. T. J. Am. Chem. SOC. 1994,116,5467. (9) Ladner, J. E.; Svensson, L. A,; Wladkowski, B. D.; Sjolin, L.; Gilliland, G. L. Work in progress. (10) (a) Binkley, J. S.; Pople, J. A,; Hehre, W. J. J. Am. Chem. SOC. 1980, 102, 939. (b) Gordon, M. S.; Binkley, J. S.; Pople, J. A,; Pietro, W. J.; Hehre, W. J. J . Am. Chem. SOC. 1982, 104, 2797. (c) Pietro, W. J.; Francl, M. M.; Hehre, W. J.; Defrees, D. J.; Pople, J. A,; Binkley, J. S. J . Am. Chem. SOC. 1982, 104, 5039. (11) All computations were performed on IBM RS/6000 workstations at CARB running the Gaussian 92 package: Frisch, M. J.; Trucks, G.W.; Head-Gordon, M.; Gill, P. M. W.; Wong, M. W.; Foresman, J. B.; Johnson, B. G.;Schlegel, H. B.; Robb, M. A.; Replogle, E. S.; Gomperts, R.; Andres, J. L.; Raghavachari, K.; Binkley, J. S.; Gonzalez, C.; Martin, R. L.; Fox, D. J.; Defrees, D. J.; Baker, J.; Stewart, J. J. P.; Pople, J. A. Gaussian 92, Revision A; Gaussian, Inc.: Pittsburgh, PA, 1992. (12) Wladkowski, B. D.; Krauss, M.; Stevens, W. J. J . Phys. Chem., in press. (13) The pK, of Lys-41 and His-12 have been estimated to be 8.6-9.1 and 5.8-6.0 respectively, for the native enzyme without bound substrate. Therefore, protonated Lys-41 and unprotonated His-12 would be expected to be the most stable protonation states at neutral pH. In the gas phase, however, the reverse is true. The gas-phase proton affinity of imidazole e) is greater than alkylamines (methylamine). Therefore, es of R1 and R2 are consistent with the limited model. To what extent desolvation of the enzyme active site shifts the results from a fully solvated pK, representation to the gas-phase proton affinity ordering remains uncertain. (14) It should be made clear that other possible orientations of the substrate within the pseudoactive site are possible which may give rise to a more stable arrangement. Structures R1 and R2 represent those relevant to the reaction channels in Figure 2, (15) (a) Gerlt, J. A,; Gassman, P, G. Submitted for publication. (b) Gerlt, J. A.; Gassman, P. G. J. Am. Chem. SOC. 1993, 115, 11552. JP9505237
