Comparison of enzyme polarization of ligands and charge-transfer effects for dihydrofolate reductase using point-charge embedded ab initio quantum mechanical and linear-scaling semiempirical quantum mechanical methods

Using quantum mechanical (QM) methods, we investigated the dependence of a number of factors on the polarization by the enzyme dihydrofolate reductase (DHFR) of its ligands-the substrates, folate and dihydrofolate, and the co factor NADPH-and evaluated the implications for facilitation of the enzymic reductions. Two quite different levels of QM description of the biomolecul ar system were used. State-of-the-art nh initio QM calculations of the liga nds were performed with the bulk DHFR environment modeled using atom-center ed point charges. At the other extreme, semiempirical AM1 QM calculations u sing the linear-scaling Mozyme formalism incorporated in MOPAC2000 allowed for consistent treatment of the 3000-atom system of both enzyme and bound l igands. The study considered the effects of a number of factors on the pola rization, including: (i) different levels of nb initio QM treatment HF, MP2 , DFT) and basis sets; (ii) different sets of molecular mechanics (MM) poin t charges in representing the bulk enzyme; (iii) inclusion of the bulk enzy me environment as either point charges in the nb initio calculations, or ex plicitly in the semiempirical calculations; (iv) ab initio QM calculations of substrate and Ligand together (combined system) or separately (noncombin ed system); (sr) degree of charge transfer between substrate and cofactor, and, for the semiempirical calculations, between bound Ligands and enzyme; (vi) polarization of the enzyme in the semiempirical calculations; (vii) di fferences in the behavior of folate and dihydrofolate; and (viii) DHFRs fro m different species (E. coli and human) and different X-ray structure coord inate sets from the same species. Polarization was analyzed mainly by diffe rences in point-charge distributions between gas-phase and bound ligands at the level of complete ligands, subcomponents of Ligands (residues), and in dividual atoms in the pterin and nicotinamide rings involved in the DHFR re actions, but some electron density differences were also calculated. Consis tent with our preliminary study (Greatbanks et al., Proteins 1999, 37, 157) , and earlier work by Bajorath et al. (Proteins 1991, 9, 217; 11, 263) for noncombined ligand systems, the DFT calculations showed an unrealistically large dipolar character for individual ligands compared with HF and MP2 res ults and anomalously large charge transfer to folate from NADPH in combined calculations, which were not shown by the HF or AM1 results. The origin of this behavior is in the representation of the gas-phase anions (the substr ates are dianionic and NADPH is L tetraanioinic), with the point-charge enz yme-embedded calculations showing polarization similar to the HF results. T he analysis highlights that successful modeling of the polarization propert ies depends on accurate representation of both the gas-phase and enzyme-bou nd electronic structures of the QM region. For both folate and dihydrofolat e at the HF and MP2 levels, changes in density from enzyme binding in the r egion of the reducible bonds (N8-C7 for folate, C6-N5 for dihydrofolate) is small, with the bulk of the polarization taking place in the N2-C2-N3 regi on near the Asp27 (or Glu30) active-site group. Polarization at C7 for fola te and C6 for dihydrofolate is negative (i.e., does not favor hydride-ion t ransfer), whereas the trend at N8 for folate, but not at N5 for dihydrofola te, favors protonation. For the Mozyme results, the substrate pterin-ring p olarization trends are similar, and also with negligible charge transfer to NADPH, and only a very small charge transfer to the enzyme. For NADPH, the HF, MP2, and Moyme results indicate charge polarization on b inding to the enzyme at the active carbon (C4) of the nicotinamide ring is favorable for hydride-ion transfer (i.e., slightly more negative), with the active hydrogen (H4) also being more negative for the HF and MP2 results. For all methodologies, the nonactive hydrogen (H4 ') becomes significantly more positive, which would reduce its potential for transfer. The Mozyme re sults show a net loss to the enzyme of similar to 0.3 electrons, mostly fro m NADPH, which is strongly localized in the vicinity of the substrate gluta mate and NADPH diphosphate and 3 '-phosphate groups. (C) 2000 John Wiley & Sons, Inc.