Native and nonnative reactions in ethanolamine ammonia-lyase are actuated by different dynamics.

We address the contribution of select classes of solvent-coupled configurational fluctuations to the complex choreography involved in configurational and chemical steps in an enzyme by comparing native and nonnative reactions conducted at different protein internal sites. The low temperature, first-order kinetics of covalent bond rearrangement of the cryotrapped substrate radical in coenzyme B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica display a kink, or increase in slope, of the Arrhenius plot with decreasing temperature. The event is associated with quenching of a select class of reaction-actuating collective fluctuations in the protein hydration layer. For comparison, a nonnative, radical reaction of the protein interior cysteine sulfhydryl group with hydrogen peroxide (H2O2) is introduced by cryotrapping EAL in an aqueous H2O2 eutectic system. The low-temperature aqueous H2O2 protein hydration and mesodomain solvent phases surrounding cryotrapped EAL are characterized by using TEMPOL spin probe electron paramagnetic resonance spectroscopy, including a freezing transition of the eutectic phase that orders the protein hydration layer. Kinetics of the cysteine-H2O2 reaction in the EAL protein interior are monitored by DEPMPO spin trapping of hydroxyl radical product. In contrast to the native reaction, the linear Arrhenius relation for the nonnative cysteine-H2O2 reaction is maintained through the solvent-protein ordering transition. The nonnative reaction is coupled to the generic local, incremental fluctuations that are intrinsic to globular proteins. The comparative approach supports the proposal that select coupled solvent-protein configurational fluctuations actuate the native reaction, and suggests that select dynamical coupling contributes to the degree of catalysis in enzymes.

Reactivity Tracking of an Enzyme Progress Coordinate.

The reactivity of individual solvent-coupled protein configurations is used to track and resolve the progress coordinate for the core reaction sequence of substrate radical rearrangement and hydrogen atom transfer in the ethanolamine ammonia-lyase (EAL) enzyme from Salmonella enterica. The first-order decay of the substrate radical intermediate is the monitored reaction. Heterogeneous confinement from sucrose hydrates in the mesophase solvent surrounding the cryotrapped protein introduces distributed kinetics in the non-native decay of the substrate radical pair capture substate, which arise from an ensemble of configurational microstates. Reaction rates increase by >103-fold across the distribution to approach that for the native enabled substate for radical rearrangement, which reacts with monotonic kinetics. The native progress coordinate thus involves a collapse of the configuration space to generate optimized reactivity. Reactivity tracking reveals fundamental features of solvent-protein-reaction configurational coupling and leads to a model that refines the ensemble paradigm of enzyme catalysis for strongly adiabatic chemical steps.

Confinement dependence of protein-associated solvent dynamics around different classes of proteins, from the EPR spin probe perspective.

Protein function is modulated by coupled solvent fluctuations, subject to the degree of confinement from the surroundings. To identify universal features of the external confinement effect, the temperature dependence of the dynamics of protein-associated solvent over 200-265 K for proteins representative of different classes and sizes is characterized by using the rotational correlation time (detection bandwidth, 10-10-10-7 s) of the electron paramagnetic resonance (EPR, X-band) spin probe, TEMPOL, which is restricted to regions vicinal to protein in frozen aqueous solution. Weak (protein surrounded by aqueous-dimethylsulfoxide cryosolvent mesodomain) and strong (no added crysolvent) conditions of ice boundary confinement are imposed. The panel of soluble proteins represents large and small oligomeric (ethanolamine ammonia-lyase, 488 kDa; streptavidin, 52.8 kDa) and monomeric (myoglobin, 16.7 kDa) globular proteins, an intrinsically disordered protein (IDP, ß-casein, 24.0 kDa), an unstructured peptide (protamine, 4.38 kDa) and a small peptide with partial backbone order (amyloid-ß residues 1-16, 1.96 kDa). Expanded and condensate structures of ß-casein and protamine are resolved by the spin probe under weak and strong confinement, respectively. At each confinement condition, the soluble globular proteins display common T-dependences of rotational correlation times and normalized weights, for two mobility components, protein-associated domain, PAD, and surrounding mesodomain. Strong confinement induces a detectable PAD component and emulation of globular protein T-dependence by the amyloid-ß peptide. Confinement uniformly impacts soluble globular protein PAD dynamics, and is therefore a generic control parameter for modulation of soluble globular protein function.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12 -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

Acinetobacter baumannii Catabolizes Ethanolamine in the Absence of a Metabolosome and Converts Cobinamide into Adenosylated Cobamides.

Acinetobacter baumannii is an opportunistic pathogen typically associated with hospital-acquired infections. Our understanding of the metabolism and physiology of A. baumannii is limited. Here, we report that A. baumannii uses ethanolamine (EA) as the sole source of nitrogen and can use this aminoalcohol as a source of carbon and energy if the expression of the eutBC genes encoding ethanolamine ammonia-lyase (EAL) is increased. A strain with an ISAba1 element upstream of the eutBC genes efficiently used EA as a carbon and energy source. The A. baumannii EAL (AbEAL) enzyme supported the growth of a strain of Salmonella lacking the entire eut operon. Remarkably, the growth of the above-mentioned Salmonella strain did not require the metabolosome, the reactivase EutA enzyme, the EutE acetaldehyde dehydrogenase, or the addition of glutathione to the medium. Transmission electron micrographs showed that when Acinetobacter baumannii or Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 synthesized AbEAL, the protein localized to the cell membrane. We also report that the A. baumannii genome encodes all of the enzymes needed for the assembly of the nucleotide loop of cobamides and that it uses these enzymes to synthesize different cobamides from the precursor cobinamide and several nucleobases. In the absence of exogenous nucleobases, the most abundant cobamide produced by A. baumannii was cobalamin. IMPORTANCE Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water. A. baumannii is an opportunistic human pathogen, considered by the CDC to be a serious threat to human health due to the multidrug resistance commonly associated with this bacterium. Knowledge of the metabolic capabilities of A. baumannii is limited. The importance of the work reported here lies in the identification of ethanolamine catabolism occurring in the absence of a metabolosome structure. In other bacteria, this structure protects the cell against damage by acetaldehyde generated by the deamination of ethanolamine. In addition, the ethanolamine ammonia-lyase (EAL) enzyme of this bacterium is unique in that it does not require a reactivase enzyme to remain active. Importantly, we also demonstrate that the A. baumannii genome encodes the functions needed to assemble adenosylcobamide, the coenzyme of EAL, from the precursor cobinamide.

Localization and interaction studies of the Salmonella enterica ethanolamine ammonia-lyase (EutBC), its reactivase (EutA), and the EutT corrinoid adenosyltransferase.

Some prokaryotes compartmentalize select metabolic capabilities. Salmonella enterica subspecies enterica serovar Typhimurium LT2 (hereafter S. Typhimurium) catabolizes ethanolamine (EA) within a proteinaceous compartment that we refer to as the ethanolamine utilization (Eut) metabolosome. EA catabolism is initiated by the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL), which deaminates EA via an adenosyl radical mechanism to yield acetaldehyde plus ammonia. This adenosyl radical can be quenched, requiring the replacement of AdoCbl by the ATP-dependent EutA reactivase. During growth on ethanolamine, S. Typhimurium synthesizes AdoCbl from cobalamin (Cbl) using the ATP:Co(I)rrinoid adenosyltransferase (ACAT) EutT. It is known that EAL localizes to the metabolosome, however, prior to this work, it was unclear where EutA and EutT localized, and whether they interacted with EAL. Here, we provide evidence that EAL, EutA, and EutT localize to the Eut metabolosome, and that EutA interacts directly with EAL. We did not observe interactions between EutT and EAL nor between EutT and the EutA/EAL complex. However, growth phenotypes of a ΔeutT mutant strain show that EutT is critical for efficient ethanolamine catabolism. This work provides a preliminary understanding of the dynamics of AdoCbl synthesis and its uses within the Eut metabolosome.

Resolution and characterization of contributions of select protein and coupled solvent configurational fluctuations to radical rearrangement catalysis in coenzyme B12-dependent ethanolamine ammonia-lyase.

Coenzyme B12 (adenosylcobalamin) -dependent ethanolamine ammonia-lyase (EAL) is the signature enzyme in ethanolamine utilization metabolism associated with microbiome homeostasis and disease conditions in the human gut. The enzyme conducts a complex choreography of bond-making/bond-breaking steps that rearrange substrate to products through a radical mechanism, with themes common to other coenzyme B12-dependent and radical enzymes. The methods presented are targeted to test the hypothesis that particular, select protein and coupled solvent configurational fluctuations contribute to enzyme function. The general approach is to correlate enzyme function with an introduced perturbation that alters the properties (for example, degree of concertedness, or collectiveness) of protein and coupled solvent dynamics. Methods for sample preparation and low-temperature kinetic measurements by using temperature-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy are detailed. A framework for interpretation of results obtained in ensemble systems under conditions of statistical equilibrium within the reacting, globally unstable state is presented. The temperature-dependence of the first-order rate constants for decay of the cryotrapped paramagnetic substrate radical state in EAL, through the chemical step of radical rearrangement, displays a piecewise-continuous Arrhenius dependence from 203 to 295K, punctuated by a kinetic bifurcation over 219-220K. The results reveal the obligatory contribution of a class of select collective protein and coupled solvent fluctuations to the interconversion of two resolved, sequential configurational substates, on the decay time scale. The select class of collective fluctuations also contributes to the chemical step. The methods and analysis are generally applicable to other coenzyme B12-dependent and related radical enzymes.

Reactivating chaperones for coenzyme B12-dependent diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

Coenzyme B12-dependent eliminases: Diol and glycerol dehydratases and ethanolamine ammonia-lyase.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.

Coupling of ethanolamine ammonia-lyase protein and solvent dynamics characterized by the temperature-dependence of EPR spin probe mobility and dielectric permittivity.

Electron paramagnetic resonance (EPR) spectroscopy is used to address the remarkable persistence of the native Arrhenius dependence of the 2-aminopropanol substrate radical rearrangement reaction in B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium from physiological to cryogenic (220 K) temperatures. Two-component TEMPOL spin probe mobility in the presence of 10 mM (0.08% v/v) 2-aminopropanol over 200-265 K demonstrates characteristic concentric aqueous-cosolvent mesodomain and protein-associated domain (PAD, hydration layer) solvent phases around EAL in the frozen solution. The mesodomain formed by the relatively small amount of 2-aminopropanol is highly confined, as shown by an elevated temperature for the order-disorder transition (ODT) in the PAD (230-235 K) and large activation energy for TEMPOL rotation. Addition of 2% v/v dimethylsulfoxide expands the mesodomain, partially relieves PAD confinement, and leads to an ODT at 205-210 K. The ODT is also manifested as a deviation of the temperature-dependence of the EPR amplitude of cob(II)alamin and the substrate radical, bound in the enzyme active site, from Curie law behavior. This is attributed to an increase in sample dielectric permittivity above the ODT at the microwave frequency of 9.5 GHz. The relatively high frequency dielectric response indicates an origin in coupled protein surface group-water fluctuations of the Johari-Goldstein ß type that span spatial scales of ∼0.1-10 Å on temporal scales of 10-10-10-7 s. The orthogonal EPR spin probe rotational mobility and solvent dielectric measurements characterize features of EAL protein-solvent dynamical coupling and reveal that excess substrate acts as a fluidizing cryosolvent to enable native enzyme reactivity at cryogenic temperatures.

Deuterium Kinetic Isotope Effects Resolve Low-Temperature Substrate Radical Reaction Pathways and Steps in B12-Dependent Ethanolamine Ammonia-Lyase.

The first-order reaction kinetics of the cryotrapped 1,1,2,2-2H4-aminoethanol substrate radical intermediate state in the adenosylcobalamin (B12)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica serovar Typhimurium are measured over the range of 203-225 K by using time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The studies target the fundamental understanding of the mechanism of EAL, the signature enzyme in ethanolamine utilization metabolism associated with microbiome homeostasis and disease conditions in the human gut. Incorporation of 2H into the hydrogen transfer that follows the substrate radical rearrangement step in the substrate radical decay reaction sequence leads to an observed 1H/2H isotope effect of approximately 2 that preserves, with high fidelity, the idiosyncratic piecewise pattern of rate constant versus inverse temperature dependence that was previously reported for the 1H-labeled substrate, including a monoexponential regime (T ≥ 220 K) and two distinct biexponential regimes (T = 203-219 K). In the global kinetic model, reaction at ≥220 K proceeds from the substrate radical macrostate, S•, and at 203-219 K along parallel pathways from the two sequential microstates, S1• and S2•, that are distinguished by different protein configurations. Decay from S•, or S1• and S2•, is rate-determined by radical rearrangement (1H) or by contributions from both radical rearrangement and hydrogen transfer (2H). Non-native direct decay to products from S1• is a consequence of the free energy barrier to the native S1• → S2• protein configurational transition. At physiological temperatures, this is averted by the fast protein configurational dynamics that guide the S1• → S2• transition.

Control of Solvent Dynamics around the B12-Dependent Ethanolamine Ammonia-Lyase Enzyme in Frozen Aqueous Solution by Using Dimethyl Sulfoxide Modulation of Mesodomain Volume.

The temperature-dependent structure and dynamics of two concentric solvent phases, the protein-associated domain (PAD) and the mesodomain, that surround the ethanolamine ammonia-lyase (EAL) protein from Salmonella typhimurium in frozen polycrystalline aqueous solution are addressed by using electron paramagnetic resonance spectroscopy of the paramagnetic nitroxide spin probe, TEMPOL, over the temperature ( T) range 190-265 K. Dimethyl sulfoxide (DMSO), added at 0.5, 2.0, and 4.0% v/v and present at the maximum freeze concentration at T ≤ 245 K, varies the volume of the interstitial aqueous DMSO mesodomain ( Vmeso) relative to a fixed PAD volume ( VPAD). The increase in Vmeso/ VPAD from 0.8 to 6.0 is quantified by the partitioning of TEMPOL between the two phases. As Vmeso/ VPAD is increased, the Arrhenius parameters for activated TEMPOL rotational motion in the mesodomain remain uniform, whereas the parameters for TEMPOL in the PAD show a progressive transformation toward the mesodomain values (higher mobility). An order-disorder transition (ODT) in the PAD is detected by the exclusion of TEMPOL from the PAD into the mesodomain. The ODT T value is systematically lowered by increased Vmeso/ VPAD (from 215 to 200 K), and PAD ordering kinks the mesodomain Arrhenius dependence. Thus there is reciprocity in PAD-mesodomain solvent coupling. The results are interpreted as a dominant influence of ice-boundary confinement on the PAD solvent structure and dynamics, which is transmitted through the mesodomain and which decreases with mesodomain volume at increased added DMSO. The systematic tuning of PAD and mesodomain solvent dynamics by the variation of added DMSO is an incisive approach for the resolution of contributions of protein-solvent dynamical coupling to EAL catalysis.

Can photolysis of the CoC bond in coenzyme B12-dependent enzymes be used to mimic the native reaction?

Coenzyme B12 (Adenosylcobalamin = AdoCbl)-dependent enzymes catalyze complex molecular transformations where cleavage of the CoC bond initiates the catalytic cycle. Alternatively, the CoC bond can be cleaved with light. In both cases, rupture of the CoC bond results in the formation of Co(II)/Ado radical pair (RP). Within the field of B12 chemistry, there has been a suspicion that photolytic cleavage can be used as a probe or a direct comparison of the native reaction. Herein, we seek to resolve what the connection between light induced RP formation and the native catalytic cycle is. We used a combined QM/MM approach to construct PESs for AdoCbl-dependent ethanolamine ammonia-lyase (EAL) as a function of axial bonds to describe the reaction mechanism. We have found that there is no direct comparison that can be made between photolysis and enzymatic cleavage as the mechanism associated with these involves different electronic states. With that being said, we have explored an alternate hypothesis for the connection which involves the one-electron reduced form of the AdoCbl cofactor. This hypothesis is in line with the concept based on proton-coupled electron transfer (PCET), which involves the formation of AdoCbl cofactor-tyrosine diradical complex. The topology of the PES for the one-electron reduced (D1) cofactor is very similar to the PES associated with photo-induced cleavage (S1). Both surfaces contain two energy minima that are, similarly, the result of two distinct electronic states. Thus, it appears that the reaction mechanism associated with the D1 surface and the S1 surface are very similar, providing a plausible connection between photolysis and native catalysis.

Protein Configurational States Guide Radical Rearrangement Catalysis in Ethanolamine Ammonia-Lyase.

The adenosylcobalamin- (coenzyme B12) dependent ethanolamine ammonia-lyase (EAL) plays a key role in aminoethanol metabolism, associated with microbiome homeostasis and Salmonella- and Escherichia coli-induced disease conditions in the human gut. To gain molecular insight into these processes toward development of potential therapeutic targets, reactions of the cryotrapped (S)-2-aminopropanol substrate radical EAL from Salmonella typhimurium are addressed over a temperature (T) range of 220-250 K by using T-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The observed substrate radical reaction kinetics are characterized by two pairs of biexponential processes: native decay to diamagnetic products and growth of a non-native radical species and Co(II) in cobalamin. The multicomponent low-T kinetics are simulated by using a minimal model, in which the substrate-radical macrostate, S⋅, is partitioned by a free-energy barrier into two sequential microstates: 1) S1⋅, a relatively high-entropy/high-enthalpy microstate with a protein configuration that captures the nascent substrate radical in the terminal step of radical-pair separation; and 2) S2⋅, a relatively low-enthalpy/low-entropy microstate with a protein configuration that enables the rearrangement reaction. The non-native, destructive reaction of S1⋅ at T ≤ 250 K is caused by a prolonged lifetime in the substrate-radical capture state. Monotonic S⋅ decay over 278-300 K indicates that the free-energy barrier to S1⋅ and S2⋅ interconversion is latent at physiological T-values. Overall, the low-temperature studies reveal two protein-configuration microstates and connecting protein-configurational transitions that specialize the S⋅ macrostate for the dual functional roles of radical capture and rearrangement enabling. The identification of new, to our knowledge, intermediate states and specific protein-fluctuation contributions to the reaction coordinate represent an advance toward development of novel therapeutic targets in EAL.

Ethanolamine is a valuable nutrient source that impacts Clostridium difficile pathogenesis.

Clostridium (Clostridioides) difficile is a gastrointestinal pathogen that colonizes the intestinal tract of mammals and can cause severe diarrheal disease. Although C. difficile growth is confined to the intestinal tract, our understanding of the specific metabolites and host factors that are important for the growth of the bacterium is limited. In other enteric pathogens, the membrane-derived metabolite, ethanolamine (EA), is utilized as a nutrient source and can function as a signal to initiate the production of virulence factors. In this study, we investigated the effects of ethanolamine and the role of the predicted ethanolamine gene cluster (CD1907-CD1925) on C. difficile growth. Using targeted mutagenesis, we disrupted genes within the eut cluster and assessed their roles in ethanolamine utilization, and the impact of eut disruption on the outcome of infection in a hamster model of disease. Our results indicate that the eut gene cluster is required for the growth of C. difficile on ethanolamine as a primary nutrient source. Further, the inability to utilize ethanolamine resulted in greater virulence and a shorter time to morbidity in the animal model. Overall, these data suggest that ethanolamine is an important nutrient source within the host and that, in contrast to other intestinal pathogens, the metabolism of ethanolamine by C. difficile can delay the onset of disease.

Electron spin-labelling of the EutC subunit in B12-dependent ethanolamine ammonia-lyase reveals dynamics and a two-state conformational equilibrium in the N-terminal, signal-sequence-associated domain.

The B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine utilisation (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Toward elucidation of the molecular basis of EAL catalysis, and its intracellular trafficking and targeting to the Eut biomicrocompartment (BMC), we have applied electron spin-labelling and electron paramagnetic resonance spectroscopy to wild-type (wt) EAL from Salmonella typhimurium, by using the sulphydryl-specific, 4-maleimido-TEMPO (4MT) spin label. One cysteine residue per active site displays exceptional reactivity with 4MT. This site is identified as ßC37 on the EutC subunit, by using 4MT-labeling of site-specific cysteine-to-alanine mutants, enzyme kinetics, and accessible surface area calculations. Electron paramagnetic resonance (EPR) spectra of 4MT-labelled wt EAL are collected over 200-265 K in frozen, polycrystalline water-only, and 1% v/v DMSO solvents. EPR simulations reveal two mobility components for each condition. Detectable spin probe reorientational motion of the two components occurs at 215 and 225 K with 1% v/v DMSO, relative to the water-only condition, consistent with formation of an aqueous-DMSO solvent mesodomain around EAL. Parallel trends in fast- and slow-reorientational correlation times and interconversion of the two populations with increasing temperature, indicate 4MT labelling of a single site (ßC37). A two-state model is proposed, in which the fast and slow motional populations represent EAL-bound and free conformations of the EutC N-terminal domain. The approximately equal proportion of each state may represent a balance between EutC and EAL protein stability and efficient targeting to the BMC.

Mesodomain and Protein-Associated Solvent Phases with Temperature-Tunable (200-265 K) Dynamics Surround Ethanolamine Ammonia-Lyase in Globally Polycrystalline Aqueous Solution Containing Dimethyl Sulfoxide.

Electron paramagnetic resonance spectroscopy of the spin probe, TEMPOL, is used to resolve solvent phases that surround the ethanolamine ammonia-lyase (EAL) protein from Salmonella typhimurium at low temperature (T) in frozen, globally polycrystalline aqueous solution and to report on the T dependence of their detectably rigid and fluid states. EAL plays a role in human gut microbiome-based disease conditions, and physicochemical studies provide insight into protein structure and mechanism, toward potential therapeutics. Temperature dependences of the rotational correlation times (τc; detection range, 10-11 ≤ τc ≤ 10-7 s) and the corresponding weights of TEMPOL tumbling components from 200 to 265 K in the presence of EAL are measured in two frozen systems: (1) water-only and (2) 1% v/v dimethyl sulfoxide (DMSO). In the water-only condition, a protein-vicinal solvent component detectably fluidizes at 230 K and melts the surrounding ice-crystalline region with increasing T, creating a bounded, relatively high-viscosity aqueous solvent domain, up to 265 K. In the EAL, 1% v/v DMSO condition, two distinct concentric solvent phases are resolved around EAL: protein-associated domain (PAD) and mesodomain. The DMSO aqueous mesodomain fluidizes at 200 K, followed by PAD fluidization at 210 K. The interphase dynamical coupling is consistent with the spatial arrangement and significant contact areas of the phases, indicated by the experimentally determined mean volume ratio, V(mesodomain)/V(PAD)/V(protein) = 0.5:0.3:1.0. The results provide a rationale for native chemical reactions of EAL at T < 250 K and an advance toward precise control of solvent dynamics as a tunable parameter for quantifying the coupling between solvent and protein fluctuations and chemical reaction steps in EAL and other enzymes.

Two Dynamical Regimes of the Substrate Radical Rearrangement Reaction in B12-Dependent Ethanolamine Ammonia-Lyase Resolve Contributions of Native Protein Configurations and Collective Configurational Fluctuations to Catalysis.

The kinetics of the substrate radical rearrangement reaction step in B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium are measured over a 92 K temperature range. The observed first-order rate constants display a piecewise-continuous Arrhenius dependence, with linear regions over 295 → 220 K (monoexponential) and 214 → 203 K (biexponential) that are delineated by a kinetic bifurcation and kinks at 219 and 217 K, respectively. The results are interpreted by using a free energy landscape model and derived microscopic kinetic mechanism. The bifurcation and kink transitions correspond to the effective quenching of two distinct sets of native collective protein configurational fluctuations that (1) reconfigure the protein within the substrate radical free energy minimum, in a reaction-enabling step, and (2) create the protein configurations associated with the chemical step. Below 217 K, the substrate radical decay reaction persists. Increases in activation enthalpy and entropy of both the microscopic enabling and reaction steps indicate that this non-native reaction coordinate is conducted by local, incremental fluctuations. Continuity in the Arrhenius relations indicates that the same sets of protein groups and interactions mediate the rearrangement over the 295 to 203 K range, but with a repertoire of configurations below 217 K that is restricted, relative to the native configurations accessible above 219 K. The experimental features of a culled reaction step, first-order kinetic measurements, and wide room-to-cryogenic temperature range, allow the direct demonstration and kinetic characterization of protein dynamical contributions to the core adiabatic, bond-making/bond-breaking reaction in EAL.

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems.

Approaches to the resolution and characterization of individual chemical steps in enzyme catalytic sequences, by using temperatures in the cryogenic range of 190-250 K, and kinetics measured by time-resolved, full-spectrum electron paramagnetic resonance spectroscopy in fluid cryosolvent and frozen solution systems, are described. The preparation and performance of the adenosylcobalamin-dependent ethanolamine ammonia-lyase enzyme from Salmonella typhimurium in the two systems exemplifies the biochemical and spectroscopic methods. General advantages of low-temperature studies are (1) slowing of reaction steps, so that measurements can be made by using straightforward T-step kinetic methods and commercial instrumentation, (2) resolution of individual reaction steps, so that first-order kinetic analysis can be applied, and (3) accumulation of intermediates that are not detectable at room temperatures. The broad temperature range from room temperature to 190 K encompasses three regimes: (1) temperature-independent mean free energy surface (corresponding to native behavior); (2) the narrow temperature region of a glass-like transition in the protein, over which the free energy surface changes, revealing dependence of the native reaction on collective protein/solvent motions; and (3) the temperature range below the glass transition region, for which persistent reaction corresponds to nonnative, alternative reaction pathways, in the vicinity of the native configurational envelope. Representative outcomes of low-temperature kinetics studies are portrayed on Eyring and free energy surface (landscape) plots, and guidelines for interpretations are presented.

Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the ß-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small.