The importance of protonation states and proton transfer in pyridoxal 5′-phosphate

The importance of protonation states and proton transfer in pyridoxal 5′-phosphate (PLP)-chemistry can hardly be overstated. protein dynamics during the catalytic cycle. In general proteins create a chemical environment and an ensemble of conformational motions to recognize different substrates with different protonations. The study of these interactions in TRPS shows that functional groups on the reacting substrate such as the phosphoryl group pyridine nitrogen phenolic oxygen and carboxyl group of each PLP-bound intermediate play a crucial role in SU-5402 constructing an appropriate molecular interface with TRPS. In particular the protonation states of the ionizable groups on the PLP cofactor may enhance or weaken the attractions between the enzyme and substrate. In addition remodulation of the charge distribution for the intermediates may help generate a suitable environment for chemical reactions. The results of our study enhance knowledge of protonation states for several PLP intermediates and help to elucidate their effects on protein dynamics in the function of TRPS and other PLP-dependent enzymes. calculations to the analysis of enzyme catalytic mechanisms provides a new level of quantification that is reshaping bioorganic mechanistic concepts.9 The combination of isotopic labeling in ssNMR experiments with computational studies grounded in quantum theory can be tailored to a scale where only protein residues in and around the active site and atoms of the substrate need be SU-5402 considered to generate atomic level models relevant to catalysis. With this detailed chemical information about the active site in hand full structural models of the protein can be explored to determine how the position SU-5402 of a single proton can change the overall protein dynamics and further activate or inactivate enzyme catalysis. Bacterial tryptophan synthase (TRPS) an α2β2 tetrameric enzyme that catalyzes the last two steps in the synthesis of L-tryptophan serves as our model system [Fig. 1(top)]. Since the 1950s TRPS has been exploited as a paradigm for understanding the catalytic and regulatory mechanisms of enzyme complexes 10 and it has recently been implicated as a target in the development of drugs for infectious diseases and herbicides.13 14 Catalysis in the α- and β-subunits is regulated via allosteric interactions that switch the protein from an open inactive conformation to a closed active conformation.12 15 Closing of the α-subunit SU-5402 is associated with large motions in α-loop L6 (αL6 residues α179-193) that switch the subunit from a disordered (open) to an ordered (closed) state while NGF2 the open-to-closed transition of the β-subunit involves motion of the communication (COMM) domain (residues β102-189). Measurement of two distances (αL6: βH6 of the COMM domain and αL6: αL2) determines whether or not the α-subunit is considered open partially closed or closed. The β-subunit is considered closed upon formation of a salt-bridge between βArg141 and βAsp305.20 Switching between the conformations is modulated by the binding of substrates to the α-site binding of monovalent cations (MVCs) and covalent interconversions between intermediates formed at the β-site. Although both the α- and β-subunits of TRPS can have open or closed conformations catalysis likely occurs only within the closed conformations.21 Substrates and products bind and dissociate via intermediates displaying open conformations (e.g. the internal aldimine (E(Ain)) while the α-aminoacrylate and quinonoid forms have completely closed conformations. Figure 1 Overall structure and chemical reactions of tryptophan synthase (TRPS). (top) TRPS is composed of an α-subunit (purple) and β-subunit (yellow). The two ligands binding to each subunit are shown in bead representation. The open partially … The detailed interactions and driving forces that induce conformational changes in TRPS have been investigated in early crystal studies and molecular dynamics (MD) simulations.22-25 Much focus has been placed on the hydrophobic tunnel of about 25 ? that connects the two active sites in crystal structures of TRPS [Fig. 1(top)]. Indole generated by the cleavage of SU-5402 indole-3-glycerol phosphate (IGP) at the α-site is transferred via this tunnel into the β-site where the synthesis of L-Trp is completed. Substrate channeling may have many advantages over the free diffusion of reaction.