. In other words, the enzyme assumes the shapes complementary to the substrate only after the substrate is bound. On the other hand, according to the population-shift model, in the vicinity of its native state the enzyme exists in the multiple conformations at its binding site. The ligand binds selectively to an active conformation and by that means shifts the equilibrium toward the binding conformation (2, 3). Here, we explore these two very different viewpoints of the ligand binding mechanism by investigating the allosteric conformational changes of Escherichia coli adenylate kinase (AdK) in the presence and absence of a ligand.AdK is a monomeric phosphotransferase enzyme that catalyzes reversible transfer of a phosphoryl group from ATP to AMP via the reaction ATP-Mg 2ϩ ϩ AMP 7 ADP-Mg 2ϩ ϩ ADP. The structure of AdK is composed of the three main domains, the CORE (residues 1-29, 68-117, and 161-214), the ATP binding domain called the LID (residues 118-167), and the NMP binding domain called the NMP (residues 30-67) (Fig.
To dissect the effects of the nucleotide-binding and catalytic metal ions on DNA polymerase mechanisms for DNA repair and synthesis, aside from the chemical reaction, we investigate their roles in the conformational transitions between closed and open states and assembly/disassembly of the active site of polymerase beta/DNA complexes before and after the chemical reaction of nucleotide incorporation. Using dynamics simulations, we find that closing before chemical reaction requires both divalent metal ions in the active site while opening after the chemical reaction is triggered by release of the catalytic metal ion. The critical closing is stabilized by the interaction of the incoming nucleotide with conserved catalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions; without the catalytic ion, other protein residues (Arg180, Arg183, Gly189) coordinate the incomer's triphosphate group through the nucleotide-binding ion. Because we also note microionic heterogeneity near the active site, Mg(2+) and Na(+) ions can diffuse into the active site relatively rapidly, we suggest that the binding of the catalytic ion itself is not a rate-limiting conformational or overall step. However, geometric adjustments associated with functional ions and proper positioning in the active site, including subtle but systematic motions of protein side chains (e.g., Arg258), define slow or rate-limiting conformational steps that may guide fidelity mechanisms. These sequential rearrangements are likely sensitively affected when an incorrect nucleotide approaches the active site. Our suggestion that subtle and slow adjustments of the nucleotide-binding and catalytic magnesium ions help guide polymerase selection for the correct nucleotide extends descriptions of polymerase pathways and underscores the importance of the delicate conformational events both before and after the chemical reaction to polymerase efficiency and fidelity mechanisms.
With an increasing number of structural, kinetic, and modeling studies of diverse DNA polymerases in various contexts, a complex dynamical view of how atomic motions might define molecular "gates" or checkpoints that contribute to polymerase specificity and efficiency is emerging. Such atomic-level information can offer insights into rate-limiting conformational and chemical steps to help piece together mechanistic views of polymerases in action. With recent advances, modeling and dynamics simulations, subject to the well-appreciated limitations, can access transition states and transient intermediates along a reaction pathway, both conformational and chemical, and such information can help bridge the gap between experimentally determined equilibrium structures and mechanistic enzymology data. Focusing on DNA polymerase β (pol β), we present an emerging view of the geometric, energetic, and dynamic selection criteria governing insertion rate and fidelity mechanisms of DNA polymerases, as gleaned from various computational studies and based on the large body of existing kinetic and structural data. The landscape of nucleotide insertion for pol β includes conformational changes, prechemistry, and chemistry "avenues", each with a unique deterministic or stochastic pathway that includes checkpoints for selective control of nucleotide insertion efficiency. For both correct and incorrect incoming nucleotides, pol β's conformational rearrangements before chemistry include a cascade of slow and subtle side chain rearrangements, followed by active site adjustments to overcome higher chemical barriers, which include critical ionpolymerase geometries; this latter notion of a prechemistry avenue fits well with recent structural and NMR data. The chemical step involves an associative mechanism with several possibilities for the initial proton transfer and for the interaction among the active site residues and bridging water molecules. The conformational and chemical events and associated barriers define checkpoints that control enzymatic efficiency and fidelity. Understanding the nature of such active site rearrangements can facilitate interpretation of existing data and stimulate new experiments that aim to probe enzyme † This work was supported by NSF Grant MCB-0316771, NIH Grants R01 GM55164 and R01 ES012692, and the donors of the American SUPPORTING INFORMATION AVAILABLEStudy of the R258A mutation in DNA polymerase β (Appendix A) and the prechemistry avenue (Appendix B). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2007 August 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptfeatures that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.DNA polymerases are essential for maintaining genomic order during DNA replication and repair (1) and thus for the long-term survival of a species. When...
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