Protonation states and catalysis: Molecular dynamics studies of intermediates in tryptophan synthase

Abstract

The importance of protonation states and proton transfer in pyridoxal 5'-phosphate (PLP)-chemistry can hardly be overstated. Although experimental approaches to investigate pKa values can provide general guidance for assigning proton locations, only static pictures of the chemical species are available. To obtain the overall protein dynamics for the interpretation of detailed enzyme catalysis in this study, guided by information from solid-state NMR, we performed molecular dynamics (MD) simulations for the PLP-dependent enzyme tryptophan synthase (TRPS), whose catalytic mechanism features multiple quasi-stable intermediates. The primary objective of this work is to elucidate how the position of a single proton on the reacting substrate affects local and global 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 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.

Direct dynamic nuclear polarization of 15N and 13C spins at 14.1 T using a trityl radical and magic angle spinning

“…U- 15 N tryptophan synthase (TS) was expressed and purified as previously described [54,55]. TS microcrystals were crystallized by 1:1 dilution of the soluble protein with 50 mM bicine in D 2 O, pH adjusted to 7.8 with CsOH, containing 14% PEG-8000 (W/V) and 3 mM spermine.…”

Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water

“…[58] We performed MD simulations using Amber14,[59] with the Amber 99SB force field for the protein and general amber force field (GAFF) for the ligands. First, we checked the protonation state using the MCCE program.…”

Human Serine Racemase: Key Residues/Active Site Motifs and Their Relation to Enzyme Function

“…Catalysis might proceed (i) in a concerted fashion without buildup of negative charge in an intermediate or transition state, (ii) via a more localized carbanionic intermediate in which the charge is not delocalized significantly into the aromatic π-system of the PLP ring, or (iii) via a fully delocalized quinonoid intermediate with a lifetime that is too short to observe with typical stopped flow instruments. In fact, collaborative work by Dunn and Mueller, utilizing a combination of NMR, X-ray crystallography and computational modeling (Caulkins et al, 2016 ; Huang et al, 2016 ), provides evidence for the intermediate case just described; namely, for the formation of such a localized “carbanionic intermediate.” As is shown in Figure 7A , this non-planar intermediate is thought to distribute electron density across the Cα-N-C4'-azallylic system rather than into the pyridine π-system. The active site lysine ε-ammonium ion is seen in close enough proximity to electrostatically stabilize this “carbanionic intermediate.”…”