Transition State Analog Complex Research Articles

Synthesis of a novel fluorinated phosphonyl C-glycoside, (3-deoxy-3-fluoro-β-d-glucopyranosyl)methylphosphonate, a potential inhibitor of β-phosphoglucomutase

β-phosphoglucomutase (βPGM) catalyzes the conversion of β-glucose 1-phosphate (βG1P) to glucose-6-phosphate (G6P), a universal source of cellular energy, in a two-step process. Transition state analogue (TSA) complexes formed from substrate analogues and a metal fluoride (MgF3− and AlF4−) enable analysis of each of these enzymatic steps independently. Novel substrate analogues incorporating fluorine offer opportunities to interrogate the enzyme mechanism using 19F NMR spectroscopy. Herein, the synthesis of a novel fluorinated phosphonyl C-glycoside (3-deoxy-3-fluoro-β-d-glucopyranosyl)methylphosphonate (1), in 12 steps (0.85 % overall yield) is disclosed. A four-stage synthetic strategy was employed, involving: 1) fluorine addition to the monosaccharide, 2) selective anomeric deprotection, 3) phosphonylation of the anomeric centre, and 4) global deprotection. Analysis of βPGM and 1 will be reported in due course.

Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase

Conformational dynamics is important for enzyme catalysis. However, engineering dynamics to achieve a higher catalytic efficiency is still challenging. In this work, we develop a new strategy to improve the activity of yeast cytosine deaminase (yCD) by engineering its conformational dynamics. Specifically, we increase the dynamics of the yCD C-terminal helix, an active site lid that controls the product release. The C-terminal is extended by a dynamical single α-helix (SAH), which improves the product release rate by up to ~8-fold, and the overall catalytic rate kcat by up to ~2-fold. It is also shown that the kcat increase is due to the favorable activation entropy change. The NMR H/D exchange data indicate that the conformational dynamics of the transition state analog complex increases as the helix is extended, elucidating the origin of the enhanced catalytic entropy. This study highlights a novel dynamics engineering strategy that can accelerate the overall catalysis through the entropy-driven mechanism.

The Relationship between Enzyme Conformational Change, Proton Transfer, and Phosphoryl Transfer in β-Phosphoglucomutase

Molecular details for the timing and role of proton transfer in phosphoryl transfer reactions are poorly understood. Here, we have combined QM models, experimental NMR measurements, and X-ray structures to establish that the transition of an archetypal phosphoryl transfer enzyme, βPGM, from a very closed near-attack conformation to a fully closed transition state analogue (TSA) conformation triggers both partial proton transfer from the general acid–base residue to the leaving group oxygen and partial dissociation of the transferring phosphoryl group from the leaving group oxygen. Proton transfer continues but is not completed throughout the reaction path of the phosphoryl transfer with the enzyme in the TSA conformation. Moreover, using interacting quantum atoms (IQA) and relative energy gradient (REG) analysis approaches, we observed that the change in the position of the proton and the corresponding increased electrostatic repulsion between the proton and the phosphorus atom provide a stimulus for phosphoryl transfer in tandem with a reduction in the negative charge density on the leaving group oxygen atom. The agreement between solution-phase 19F NMR measurements and equivalent QM models of βPGMWT and βPGMD10N TSA complexes confirms the protonation state of G6P in the two variants, validating the employed QM models. Furthermore, QM model predictions of an AlF4 distortion in response to the proton position are confirmed using high resolution X-ray crystal structures, not only providing additional validation to the QM models but also further establishing metal fluorides as highly sensitive experimental predictors of active-site charge density distributions.

Octahedral Trifluoromagnesate, an Anomalous Metal Fluoride Species, Stabilizes the Transition State in a Biological Motor.

Isoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3– and AlF4–, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)−, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)− species, indicating the significance of this TSA for studies of biological motors.

1H, 15N, 13C backbone resonance assignments of the P146A variant of beta-phosphoglucomutase in a transition state analogue complex with glucose 6-phosphate and trifluoromagnesate (MgF3-)

1H, 15N, 13C backbone resonance assignments of the P146A variant of beta-phosphoglucomutase in a transition state analogue complex with glucose 6-phosphate and trifluoromagnesate (MgF3-)

Van der Waals Contact between Nucleophile and Transferring Phosphorus Is Insufficient To Achieve Enzyme Transition-State Architecture

Phosphate plays a crucial role in biology because of the stability of the phosphate ester bond. To overcome this inherent stability, enzymes that catalyze phosphoryl transfer reactions achieve enormous rate accelerations to operate on biologically relevant time scales, and the mechanisms that underpin catalysis have been the subject of extensive debate. In an archetypal system, β-phosphoglucomutase catalyzes the reversible isomerization of β-glucose 1-phosphate and glucose 6-phosphate via two phosphoryl transfer steps using a β-glucose 1,6-bisphosphate intermediate and a catalytic MgII ion. In the present work, a variant of β-phosphoglucomutase, where the aspartate residue that acts as a general acid–base is replaced with asparagine, traps highly stable complexes containing the β-glucose 1,6-bisphosphate intermediate in the active site. Crystal structures of these complexes show that, when the enzyme is unable to transfer a proton, the intermediate is arrested in catalysis at an initial stage of phosphoryl transfer. The nucleophilic oxygen and transferring phosphorus atoms are aligned and in van der Waals contact, yet the enzyme is less closed than in transition-state (analogue) complexes, and binding of the catalytic MgII ion is compromised. Together, these observations indicate that optimal closure and optimal MgII binding occur only at higher energy positions on the reaction trajectory, allowing the enzyme to balance efficient catalysis with product dissociation. It is also confirmed that the general acid–base ensures that mutase activity is ∼103 fold greater than phosphatase activity in β-phosphoglucomutase.

Picosecond Active‐Site Dynamics Correlate with the Temperature Dependence of KIEs in Enzyme‐Catalyzed Hydride Transfer

Picosecond time scale motions in enzymes have often been invoked to explain the temperature dependence of primary kinetic isotope effects (KIE) in enzyme‐catalyzed hydride transfer reactions, but these experiments come short of actually measuring the dynamics in these systems on timescales relevant to the making and breaking of bonds. Similar to other enzymes, wild‐type Formate dehydrogenase (FDH) from Candida boidinii exhibits a temperature independent KIE that becomes temperature dependent when space is opened up in the active site via mutation of the V123 residue to alanine and glycine. Transition state analog complexes of FDH bound to azide offer a unique opportunity to investigate how these mutations influence active site dynamics. With a combination of 2D IR spectroscopy, X‐ray crystallography, and molecular dynamic simulations, we report a comprehensive view of fast motions of the active site and their relationship to the hydride transfer. Azide bound to FDH exhibits oscillatory frequency fluctuations on the ps timescale, and the amplitude of these fluctuations increases when the active site space increases in correlation with the temperature dependence of the KIE. Both the kinetic and dynamic phenomena can be modeled computationally. These results provide the first means of directly connecting temperature dependence of KIE's to dynamic motion in the active site in an enzyme‐catalyzed reaction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

1H, 15N, 13C backbone resonance assignments of human phosphoglycerate kinase in a transition state analogue complex with ADP, 3-phosphoglycerate and magnesium trifluoride

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF3−) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO3−), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the 1H, 15N and 13C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF3:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the 1H–15N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.

Elevated μs-ms timescale backbone dynamics in the transition state analog form of arginine kinase.

Arginine kinase catalyzes reversible phosphoryl transfer between arginine and ATP. Crystal structures of arginine kinase in an open, substrate-free form and closed, transition state analog (TSA) complex indicate that the enzyme undergoes substantial domain and loop rearrangements required for substrate binding, catalysis, and product release. Nuclear magnetic resonance (NMR) has shown that substrate-free arginine kinase is rigid on the ps-ns timescale (average S2=0.84±0.08) yet quite dynamic on the µs-ms timescale (35 residues with Rex, 12%), and that movements of the N-terminal domain and the loop comprising residues I182-G209 are rate-limiting on catalysis. Here, NMR of the TSA-bound enzyme shows similar rigidity on the ps-ns timescale (average S2=0.91±0.05) and substantially increased μs-ms timescale dynamics (77 residues; 22%). Many of the residues displaying μs-ms dynamics in NMR Carr-Purcell-Meiboom-Gill (CPMG) 15N backbone relaxation dispersion experiments of the TSA complex are also dynamic in substrate-free enzyme. However, the presence of additional dynamic residues in the TSA-bound form suggests that dynamics extend through much of the C-terminal domain, which indicates that in the closed form, a larger fraction of the protein takes part in conformational transitions to the excited state(s). Conformational exchange rate constants (kex) of the TSA complex are all approximately 2500s-1, higher than any observed in the substrate-free enzyme (800-1900s-1). Elevated μs-ms timescale protein dynamics in the TSA-bound enzyme is more consistent with recently postulated catalytic networks involving multiple interconnected states at each step of the reaction, rather than a classical single stabilized transition state.

Observing enzyme ternary transition state analogue complexes by 19F NMR spectroscopy.

Ternary transition state analogue (TSA) complexes probing the isomerization of β-d-glucose 1-phosphate (G1P) into d-glucose 6-phosphate (G6P) catalyzed by catalytically active, fluorinated (5-fluorotryptophan), β-phosphoglucomutase (βPGM) have been observed directly by 19F NMR spectroscopy. In these complexes MgF3- and AlF4- are surrogates for the transferring phosphate. However, the relevance of these metal fluorides as TSA complexes has been queried. The 1D 19F spectrum of a ternary TSA complex presented a molar equivalence between fluorinated enzyme, metal fluoride and non-isomerizable fluoromethylenephosphonate substrate analogue. Ring flips of the 5-fluoroindole ring remote from the active site were observed by both 19F NMR and X-ray crystallography, but did not perturb function. This data unequivocally demonstrates that the concentration of the metal fluoride complexes is equivalent to the concentration of enzyme and ligand in the TSA complex in aqueous solution.

The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase

Arginine kinase provides a model for functional dynamics, studied through crystallography, enzymology, and nuclear magnetic resonance. Structures are now solved, at ambient temperature, for the transition state analog (TSA) complex. Analysis of quasi-rigid sub-domain displacements show that differences between the two TSA structures average about 5% of changes between substrate-free and TSA forms, and they are nearly co-linear. Small backbone hinge rotations map to sites that also flex on substrate binding. Anisotropic atomic displacement parameters (ADPs) are refined using rigid-body TLS constraints. Consistency between crystal forms shows that they reflect intrinsic molecular properties more than crystal lattice effects. In many regions, the favored directions of thermal/static displacement areappreciably correlated with movements on substrate binding. Correlation between ADPs and larger substrate-associated movements implies that the latter approximately follow paths of low-energy intrinsic motions.

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction

AbstractStates along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

MgF3- and AlF4- transition state analogue complexes of yeast phosphoglycerate kinase.

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) transition state analogue complexes and analyzed using 19F, 1H waterLOGSY and 1H chemical shift perturbation NMR spectroscopy. We observed the first 19F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.

19F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA‐Catalyzed GTP Hydrolysis

AbstractMolecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use 19F NMR chemical shifts in the MgF3− transition state analogue (TSA) complex as a spectroscopic reporter to indicate electron distribution for the γ‐PO3− oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density. This was validated by QM calculations giving a picture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the γ‐PO3− group based on the structure of a RhoA/GAP‐GDP‐MgF3− TSA complex. The TS model displays a network of 20 hydrogen bonds, including the GAP Arg85′ side chain, but neither phosphate torsional strain nor general base catalysis is evident. The nucleophilic water occupies a reactive location different from that in multiple ground state complexes, arising from reorientation of the Gln‐63 carboxamide by Arg85′ to preclude direct hydrogen bonding from water to the target γ‐PO3− group.

(19)F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA-Catalyzed GTP Hydrolysis.

Molecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use 19F NMR chemical shifts in the MgF3 − transition state analogue (TSA) complex as a spectroscopic reporter to indicate electron distribution for the γ‐PO3 − oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density. This was validated by QM calculations giving a picture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the γ‐PO3 − group based on the structure of a RhoA/GAP‐GDP‐MgF3 − TSA complex. The TS model displays a network of 20 hydrogen bonds, including the GAP Arg85′ side chain, but neither phosphate torsional strain nor general base catalysis is evident. The nucleophilic water occupies a reactive location different from that in multiple ground state complexes, arising from reorientation of the Gln‐63 carboxamide by Arg85′ to preclude direct hydrogen bonding from water to the target γ‐PO3 − group.

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction.

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-D-glucose 1-phosphate (βG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-D-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-D-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.

A crystallographic study of the Toho-1 β-lactamase acylation mechanism

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

Characterization of a putative oomycete taurocyamine kinase: Implications for the evolution of the phosphagen kinase family

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. Within animal species, these enzymes play a critical role in energy homeostasis by catalyzing the reversible transfer of a high-energy phosphoryl group from Mg⋅ATP to an acceptor molecule containing a guanidinium group. In this work, a putative PK gene was identified in the oomycete Phytophthora sojae that was predicted, based on sequence homology, to encode a multimeric hypotaurocyamine kinase. The recombinant P. sojae enzyme was purified and shown to catalyze taurocyamine phosphorylation efficiently (kcat/KM (taurocyamine) = 2 × 10(5) M(-1) s(-1)) and glycocyamine phosphorylation only weakly (kcat/KM (glycocyamine) = 2 × 10(2) M(-1) s(-1)), but lacked any observable kinase activity with the more ubiquitous guanidinium substrates, creatine or arginine. Additionally, the enzyme was observed to be dimeric but lacked cooperativity between the subunits in forming a transition state analog complex. These results suggest that protozoan PKs may exhibit more diversity in substrate specificity than was previously thought.

Backbone resonance assignments of the 42 kDa enzyme arginine kinase in the transition state analogue form

Nearly complete backbone resonance assignments for the 357 residue, 42 kDa enzyme arginine kinase in a transition state analogue (TSA) complex are presented. The TSA is a quaternary complex of arginine kinase, MgADP, arginine, and nitrate. About 93% (320 of 344) of the non-proline backbone amides were assigned using an enzyme enriched with (2)H, (13)C, and (15)N in combination with three enzyme samples prepared with a single (15)N-labeled amino acid (K, L, and R). The amide assignments will provide the foundation for investigating the dynamics of arginine kinase when in a TSA complex.