Why Do Divalent Metal Ions Either Promote or Inhibit Enzymatic Reactions?

Abstract

Divalent metal ions are essential to many enzymatic reactions involving nucleic acids, but their critical and specific role still needs to be uncovered. Restriction endonucleases are a prominent group of such metal-requiring enzymes. Large scale accurate simulations of Mg- and Ca-BamHI elucidate the mechanism of the catalytic reaction leading to DNA cleavage and show that it involves the concerted action of two metal ions and water molecules. It is also established that what is decisive for the dramatically different behavior of magnesium (a cocatalyst) and calcium (an inhibitor) are kinetic factors and not the properties of the prereactive states of the enzymes. A new perspective is opened for the understanding of the functional role of metal ions in biological processes. Divalent metal ions are essential to many enzymatic reactions involving nucleic acids, but their critical and specific role still needs to be uncovered. Restriction endonucleases are a prominent group of such metal-requiring enzymes. Large scale accurate simulations of Mg- and Ca-BamHI elucidate the mechanism of the catalytic reaction leading to DNA cleavage and show that it involves the concerted action of two metal ions and water molecules. It is also established that what is decisive for the dramatically different behavior of magnesium (a cocatalyst) and calcium (an inhibitor) are kinetic factors and not the properties of the prereactive states of the enzymes. A new perspective is opened for the understanding of the functional role of metal ions in biological processes. Many enzymatic reactions involving nucleic acids are known to critically rely on the cooperative action of divalent metal cofactors, such as magnesium, that are incorporated into the protein as positively charged ions (1Cowan J.A. Chem. Rev. 1998; 98: 1067-1087Crossref PubMed Scopus (396) Google Scholar). Although quite well established, their functional role has not yet received a satisfactory explanation and is still a matter of debate and speculation. Among these reactions, the ones more often investigated experimentally pertain to the family of enzymes called type II restriction endonucleases. These enzymes are highly site-specific; their biological function is that of cleaving DNA at both strands (2Pingoud A. Jeltsch A. Eur. J. Biochem. 1997; 246: 1-22Crossref PubMed Scopus (302) Google Scholar), and they all require metal cofactors. This is a process of primary biochemical importance. For example, this process provides protection to bacterial cells against bacteriophage infection, occurs naturally during programmed cell death (apoptosis), and has been exploited in genetic engineering. The experimental information accumulated over the years (seee.g. Refs. 1Cowan J.A. Chem. Rev. 1998; 98: 1067-1087Crossref PubMed Scopus (396) Google Scholar, 2Pingoud A. Jeltsch A. Eur. J. Biochem. 1997; 246: 1-22Crossref PubMed Scopus (302) Google Scholar, 3Horton N.C. Newberry K.J. Perona J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13489-13494Crossref PubMed Scopus (104) Google Scholar, 4Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Science. 1995; 269: 656-663Crossref PubMed Scopus (291) Google Scholar, 5Jen-Jacobson L. Biopolymers. 1997; 44: 153-180Crossref PubMed Scopus (172) Google Scholar, 6Viadiu H. Aggarwal A.K. Nat. Struct. Biol. 1998; 5: 910-916Crossref PubMed Scopus (146) Google Scholar, 7Dupureur C.M. Hallman L.M. Eur. J. Biochem. 1999; 261: 261-268Crossref PubMed Scopus (29) Google Scholar, 8Horton J.R. Cheng X. J. Mol. Biol. 2000; 300: 1049-1056Crossref PubMed Scopus (71) Google Scholar, 9Beernink P.T. Segelke B.W. Hadi M.Z. Erzberger J.P. Wilson III, D.M. Rupp B. J. Mol. Biol. 2001; 307: 1023-1034Crossref PubMed Scopus (164) Google Scholar, 10Horton N.C. Connolly B.A. Perona J.J. J. Am. Chem. Soc. 2000; 122: 3314-3324Crossref Scopus (35) Google Scholar) mostly concerns the structural properties of the protein-DNA complexes and the effect of mutations on their activity. More is needed to unravel the precise mechanism driving the cleavage of the phosphodiester bond at the DNA strands (11Galburt E.A. Stoddard B.L. Biochemistry. 2002; 41: 13851-13860Crossref PubMed Scopus (113) Google Scholar). Although there is little doubt that the reaction takes place via a concerted nucleophilic substitution, it is still unclear whether the attacking nucleophile is a water molecule or a hydroxide ion. More importantly the activity of these enzymes crucially depends on the presence and identity of the metal ions, 1Between one and three metal binding sites have been observed in the crystallographic structures of different restriction endonucleases, but the correlation between number of ions and activity is unclear. but simple features, such as ionic size, electronegativity, or average coordination number, do not simply correlate with it. Other issues concern the extent to which the characteristics of the initial state of the reaction or the presence of water molecules can affect its development. Here we make definitive progress toward answering all these questions with the aid of accurate computer simulations that provide the so far missing insight into the mechanism of the catalytic reaction and a reliable evaluation of its energetics. The validity of any computational approach to enzymatic processes critically relies on a quantum mechanical description of the active site, but the modeling often suffers from the size limitation typical of ab initio calculations. To overcome this restriction, combined quantum-classical approaches have been proposed for a long time (12Warshel A. Levitt M. J. Mol. Biol. 1976; 103: 227-249Crossref PubMed Scopus (3695) Google Scholar, 13Singh U.C. Kollman P.A. J. Comput. Chem. 1986; 7: 718-730Crossref Scopus (1069) Google Scholar, 14Field M.J. Bash P.A. Karplus M. J. Comput. Chem. 1990; 11: 700-733Crossref Scopus (2185) Google Scholar) that partition the system into two subsystems: the close environment of the reaction site, treated quantum mechanically (QM), 2The abbreviations used are: QM, quantum mechanics; MM, molecular mechanics; DFT, density functional theory; GROMOS, Groningen molecular simulation and the outer region described with a molecular mechanics model (MM). However, only recently has this method started to provide the required level of accuracy (15Gao J. Lipkowitz K.B. Boyd D.B. Reviews in Computational Chemistry. 7. VCH Publishers, New York1996: 119-185Google Scholar, 16Monard G. Merz K.M. Acc. Chem. Res. 1999; 32: 904-911Crossref Scopus (327) Google Scholar, 17Laio A. Vande Vondele J. Röthlisberger U. J. Chem. Phys. 2002; 116: 6941-6947Crossref Scopus (557) Google Scholar). Our QM/MM approach combines two well established methodologies, namely density functional theory (DFT) (18Parr R.G. Yang W. Density-Functional Theory of Atoms and Molecules. Oxford University Press, New York1989Google Scholar) and the GROMOS force field (19van Gunsteren W.F. Billeter S.R. Eising A.A. Hünenberger P.H. Krüger P. Mark A.E. Scott W.R.P. Tironi I.G. Biomolecular Simulation: The GROMOS96 Manual and User Guide. VdF Hochschulverlag AG der ETH Zürich, Zurich, Switzerland1996Google Scholar), and includes a new careful treatment of the electrostatic interactions (20Mordasini T. Curioni A. Bursi R. Andreoni W. ChemBioChem. 2003; (in press)PubMed Google Scholar). We apply this method to the study of the restriction endonucleaseBamHI (1Cowan J.A. Chem. Rev. 1998; 98: 1067-1087Crossref PubMed Scopus (396) Google Scholar, 4Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Science. 1995; 269: 656-663Crossref PubMed Scopus (291) Google Scholar, 5Jen-Jacobson L. Biopolymers. 1997; 44: 153-180Crossref PubMed Scopus (172) Google Scholar, 6Viadiu H. Aggarwal A.K. Nat. Struct. Biol. 1998; 5: 910-916Crossref PubMed Scopus (146) Google Scholar) and focus on the comparison of the activities of magnesium and calcium, which are experimentally known to act as cocatalyst and inhibitor, respectively, of the DNA cleavage reaction. This drastically different behavior of magnesium and calcium is quite common, and the reasons for it are largely unknown. The advantage that this specific system provides for a thorough investigation consists in the availability of x-ray structures that could be used as reference for our simulations: these structures refer to the complexes obtained by cocrystallizing the protein-DNA complex with calcium and with manganese 3The use of manganese is often meant to provide information on the analog case of magnesium, which is essentially invisible in x-ray measurements but is the natural, and thus most interesting, cocatalyst. and can be considered as representative of the pre- and postreactive states, respectively. In particular, by comparing the two states, the structure of the protein appeared to be largely insensitive to the reaction as evidenced by the overall root mean square displacement of only 0.33 Å between the Cα positions of the pre- and postreactive states. Two metal atoms (A and B) were observed at the active site, corroborating earlier proposals for a two-metal mechanism (4Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Science. 1995; 269: 656-663Crossref PubMed Scopus (291) Google Scholar), the nature of which will emerge clearly from our results. The QM subsystem was treated in the framework of DFT (18Parr R.G. Yang W. Density-Functional Theory of Atoms and Molecules. Oxford University Press, New York1989Google Scholar) using Becke's approximation (21Becke A.D. Phys. Rev. A. 1988; 38: 3098-3100Crossref PubMed Scopus (45699) Google Scholar) for the exchange energy functional and the Lee-Yang-Parr approximation (22Lee C. Yang W. Parr R.G. Phys. Rev. B. 1988; 37: 785-789Crossref PubMed Scopus (87769) Google Scholar) for the correlation energy functional. The QM calculations were performed with the Car-Parrinello molecular dynamics (CPMD) code 4CPMD: copyright IBM Corp. 1990, 1997, 2001 and Max-Planck-Institut für Festkörperforschung, Stuttgart, Germany, 1997; see www.cpmd.org. using a plane-wave basis set for the description of the valence electron wave functions (energy cut-off 70 Rydberg) and norm-conserving angular momentum-dependent pseudopotentials for the description of the core-valence interaction. The MM subsystem was treated within the GROMOS force field (19van Gunsteren W.F. Billeter S.R. Eising A.A. Hünenberger P.H. Krüger P. Mark A.E. Scott W.R.P. Tironi I.G. Biomolecular Simulation: The GROMOS96 Manual and User Guide. VdF Hochschulverlag AG der ETH Zürich, Zurich, Switzerland1996Google Scholar), which is well established (see e.g.Ref. 23Daura X. Gademann K. Schäfer H. Jaun B. Seebach D. van Gunsteren W.F. J. Am. Chem. Soc. 2001; 123: 2393-2404Crossref PubMed Scopus (190) Google Scholar) for the determination of both the structure and the dynamics of proteins in aqueous solutions (24Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Hermans J. Pullman B. Intermolecular Forces. Reidel, Dordrecht, The Netherlands1981: 331-342Google Scholar). The parametrization used corresponds to the 43A1 version of the force field. The interface between the two subsystems was modeled using the link-atom method (14Field M.J. Bash P.A. Karplus M. J. Comput. Chem. 1990; 11: 700-733Crossref Scopus (2185) Google Scholar). The QM subsystem was enclosed in a box (of edges 43.36, 48.55, and 39.21 atomic units) and treated as an “embedded cluster” namely using the Hockney method (25Barnett R.N. Landman U. Phys. Rev. B. 1993; 48: 2081-2097Crossref Scopus (517) Google Scholar) and accounting for the influence of the MM subsystem via explicit calculations of van der Waals and electrostatic interactions. The electrostatic potential of the MM atoms was calculated in the QM region exactly within a cut-off radius of 15 Å and including a Poisson-Boltzmann reaction field correction (26Tironi I. Sperb R. Smith P.E. van Gunsteren W.F. J. Chem. Phys. 1995; 102: 5451-5459Crossref Scopus (1178) Google Scholar) outside. The influence of the QM atoms on the MM atoms was accounted for via the electrostatic interaction. The QM region was represented by a distribution of effective charges, which we obtained from fitting to the ab initio electrostatic potential using a novel and suitable procedure that avoids common errors due to inconsistency between quantum mechanically derived parameters and the rest of the force field (20Mordasini T. Curioni A. Bursi R. Andreoni W. ChemBioChem. 2003; (in press)PubMed Google Scholar). The initial structure is taken from the x-ray structure of the calcium complex (6Viadiu H. Aggarwal A.K. Nat. Struct. Biol. 1998; 5: 910-916Crossref PubMed Scopus (146) Google Scholar) (Protein Data Bank entry 2BAM), and hydrogen atoms are added as required by the force field (19van Gunsteren W.F. Billeter S.R. Eising A.A. Hünenberger P.H. Krüger P. Mark A.E. Scott W.R.P. Tironi I.G. Biomolecular Simulation: The GROMOS96 Manual and User Guide. VdF Hochschulverlag AG der ETH Zürich, Zurich, Switzerland1996Google Scholar). The system was solvated in a rectangular box of 14,819 simple point charge water molecules (24Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Hermans J. Pullman B. Intermolecular Forces. Reidel, Dordrecht, The Netherlands1981: 331-342Google Scholar). Sodium ions were added in proximity of the DNA chain to provide charge neutralization. The optimization procedure (of the electronic and geometric variables) consisted of three consecutive steps. First the structure of the QM system was optimized keeping the positions of the MM atoms fixed, consistent with the experimental observation of a minor difference between pre- and postreactive states. Next the MM geometry was relaxed with molecular dynamics runs and optimized. The interaction with the QM system (fixed) was modeled as discussed above. Finally the structure of the QM subsystem was optimized again. Only minor changes were observed in these last two steps. In our simulations, the system is immersed in water to provide a realistic model (see Fig. 1) for the study of the enzymatic reaction. As the starting configuration, we used the crystallographic structure of the BamHI-DNA complex with calcium and optimized its structure (Fig. 2 b) in water; we then repeated this procedure after replacing calcium with magnesium (Fig. 2 a). As attacking agent, we considered a hydroxide generated either by a water molecule (4Newman M. Strzelecka T. Dorner L.F. Schildkraut I. Aggarwal A.K. Science. 1995; 269: 656-663Crossref PubMed Scopus (291) Google Scholar, 6Viadiu H. Aggarwal A.K. Nat. Struct. Biol. 1998; 5: 910-916Crossref PubMed Scopus (146) Google Scholar) deprotonated in situ (intrinsic mechanism) or by an external source (extrinsic mechanism) (27Fuxreiter M. Osman R. Biochemistry. 2001; 40: 15017-15023Crossref PubMed Scopus (14) Google Scholar). Fig. 2 refers only to the modeling of the latter reaction. In this case, only minor geometrical differences emerged in the initial states of the two enzymes apart from the expected decrease of the metal-oxygen distances (on average from 2.6 to 2.2 Å) on passing from calcium to magnesium. Water molecules are part of the metal coordination shell and are crucial to guarantee the preferred coordination number for both the A and B ions. If the hydroxide is replaced by a water molecule as attacking species, more significant differences between the A and B ion environments in the initial state as well as between those of calcium and magnesium are observed. For example, the magnesium B ion loses the phosphate O-2 oxygen as binding partner in a 5-fold coordination. However, the structural discrepancies we have detected in the prereactive states of the two metal-requiring proteins are minor and do not reflect a significantly different propensity of calcium and magnesium to donate electrons or polarize the surrounding bonding pattern. This is best illustrated by the spatial distribution of the charges obtained from the topological analysis of the electron density along the lines of Bader's theory (28Bader R.F.W. Atoms in Molecules: A Quantum Theory. Oxford University Press, New York1994Google Scholar) (Fig. 3). The atomic charges associated with the A and B ions are the same, and only a small difference is found between calcium and magnesium. Both A and B ions help stabilize a cluster distribution of negative charges localized at the active This is in with the experimental observation of an of the of the protein-DNA complex in the presence of divalent ions L. Biopolymers. 1997; 44: 153-180Crossref PubMed Scopus (172) Google Scholar). In the two-metal mechanism in H. Aggarwal A.K. Nat. Struct. Biol. 1998; 5: 910-916Crossref PubMed Scopus (146) Google Scholar, the role of the A ion is that of ions generated by the of and the of to act as cocatalyst was as to provide the of ions for the nucleophilic of an that of either or The role of the A ion is by our calculations for magnesium, but the is for our it has that the active not any in the initial state of the binding that could provide a simple explanation for the dramatically different behavior of magnesium and of the optimized in the close of the phosphate in the initial and states of the magnesium (a and calcium distances are in Large states of both magnesium and calcium atomic charges obtained using Bader's approach (28Bader R.F.W. Atoms in Molecules: A Quantum Theory. Oxford University Press, New York1994Google Scholar) are in the to this were in the for of and hydrogen atoms were is the charge distribution a the the all over the region of the active Bader's atomic charges only from the ionic charges, they are for magnesium and for Large We to the reaction and the energy For the phosphodiester cleavage by the be it or we its approach to the phosphate as reaction For the of the water we its from the in Fig. In the case of magnesium, the bond by the hydroxide in the and in the energy are obtained magnesium is replaced by calcium, namely and respectively. The presence of calcium of magnesium also to a the energy to of water in namely from to the the to a hydroxide from the to the metal coordination which is not calculated is expected to be only different in the two of the energy in this process can be obtained M. J. A. J. Am. Chem. Soc. 1995; Scopus Google Scholar) from the difference between the of the metal water and the experimental external P. Dorner L.F. R. Schildkraut I. L. J. Mol. Biol. 2001; 307: PubMed Scopus Google Scholar). This to for magnesium and for In our show that the of the reaction is decisive for the dramatically different behavior of the two divalent ions. Our calculations also show that the mechanism is the most which is consistent with model calculations of the energy in the presence of the for the of the hydroxide in the two (27Fuxreiter M. Osman R. Biochemistry. 2001; 40: 15017-15023Crossref PubMed Scopus (14) Google Scholar). energy of was derived from the of the of the phosphodiester bond cleavage reaction P. Dorner L.F. R. Schildkraut I. L. J. Mol. Biol. 2001; 307: PubMed Scopus Google Scholar) by the magnesium If we the due to the of the hydroxide in the system from we a of with our calculated of for the energy that the is A understanding of the difference in the calculated energy can be obtained by the active in the region of the (Fig. These structures show the presence of a for the with both the attacking and in This is consistent with the geometry of the state in the of phosphate E.A. J. Am. Chem. Soc. PubMed Scopus Google Scholar). Here the structural differences between the calcium and magnesium environments are more in the initial and their role in the for the to can be our simulations of the active magnesium it that the role of the B ion is crucial in the state of the In the B ion its coordination with oxygen from to This is what a water molecule to the coordination of the metal and to via hydrogen water which the of the the of the group of the oxygen coordination shell of is not so for magnesium. This is not the case for calcium, which to to a In the system is to the in a of the bonding between the phosphate and the metal the calcium B ion not its coordination but to all oxygen thus to stabilize a that be for the to As Fig. 2 the A ion also to its in a of the atoms it in to the case of the magnesium in which the A ion a structure during the reaction (see Fig. and this the energy required by the calcium complex with to the magnesium complex in the reaction to These simulations have the mechanism by which magnesium ions the in cleaving the phosphodiester bond in DNA strands and calcium ions the catalytic reaction. These have earlier (6Viadiu H. Aggarwal A.K. Nat. Struct. Biol. 1998; 5: 910-916Crossref PubMed Scopus (146) Google Scholar) that a crucial role in the reaction is by water molecules as well as by a metal the one binding the attacking agent, and their concerted of This to a state with a that the coordination shell of the metal ions in a that is with the binding of magnesium but not with that of this those for the calcium complex can be expected for complexes and which are more to magnesium and also act as (6Viadiu H. Aggarwal A.K. Nat. Struct. Biol. 1998; 5: 910-916Crossref PubMed Scopus (146) Google Scholar). from were to for the difference of the electronic structure of the two enzymes that drastically the of the reaction the properties of the prereactive states. The of this investigation is largely due to our computational which relies on a careful description of all interactions and on a size of the QM In as we have the size of the QM subsystem drastically changes the nature of the and thus the behavior of the and interactions with the MM region it The found in the of the energy of the with the two different is such that of the accuracy of these calculations an of the DFT functional or a for the will not the explanation we have for their different These also a new perspective on the of enzymatic reactions on metal ion (1Cowan J.A. Chem. Rev. 1998; 98: 1067-1087Crossref PubMed Scopus (396) Google Scholar) and an and accurate method that can be to and to specific such as reaction We are to and K. for to this