The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

Identification and characterization of single chain anti-cocaine catalytic antibodies

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.     

Interaction of Brain-Type Creatine Kinase with its Transition State Analog: Kinetics of Inhibition and Conformational Changes

Abstract

The effects of components of the transition state analog (creatine, MgADP, planar anion) on the kinetics and conformation of creatine kinase isozyme BB from monkey brain was studied. From analysis of the reaction time course using the pH stat assay, it was shown that during accumulation of the reaction products (ADP and creatine phosphate), among several anions added, nitrate proved the most effective in inhibiting catalytic activity. Maximum inhibition (77%) was achieved with 50 mM nitrate. The K_m for ATP was 0.48 mM and in the presence of 2.5 mM nitrate, 2.2 mM; for ATP in the presence of the dead-end complex, creatine and ADP, the apparent Km was 2.0 mM and theK wasO.16mM; in the presence of the transition state analog, MgADP + NO₃” + creatine, the K was estimated to be 0.04 mM.

Ultraviolet difference spectra of creatine kinase revealed significant differences only in the presence of the complete mixture of the components of the transition state analog. Comparison of gel nitration elution profiles for creatine kinase in the absence and presence of the complete mixture of components of the transition state analog did not reveal any differences in elution volume. Addition of components of the transition state analog to creatine kinase resulted in only a marginal change in intrinsic fluorescence. The presence of the components of the transition state analog increased the rate of reactivity of the enzyme with trinitrobenzenesulfonic acid from k = 6.06 ±0.05M^?1min to 6.96 ± 0.11 M^?1min^?1.

This study provides evidence that, like the muscle isozyme of creatine kinase, the brain form is effectively inhibited by the transition state analog. However, the inhibition is accompanied by small changes in the overall conformation of the protein. This adds to the evidence that the functional differences of the isozymic forms of creatine kinase cannot be attributed to differences in kinetic properties.     

Towards New Thymidine Phosphorylase/PD-ECGF Inhibitors Based on the Transition State of the Enzyme Reaction

Abstract

Computational studies have been conducted to built a closed form of TPase and to characterize the transition state of the phosphorylisis reaction catalyzed by TPase. The results obtained point to a crucial role of His-85 and the O2 of thymine in the catalysis. This modelled transition state forms the basis for the design of new TPase inhibitors.     

Computational study of a transition state analog of phosphoryl transfer in the Ras–RasGAP complex: AlF x versus MgF 3 –

The structures of the complexes between Ras•GDP bound to RasGAP in the presence of three probable γ-phosphate analogs (AlF₃, AlF₄⁻ and MgF₃⁻) for the transition state (TS) of the hydrolysis of guanosine triphosphate (GTP) by the Ras-RasGAP enzymes have been modeled by quantum mechanical—molecular mechanical (QM/MM) calculations. These simulations contribute to the dispute on the nature of the TS in the hydrolysis reaction, since medium resolution X-ray crystallography cannot discern among stereochemically similar isoelectronic species (e.g., AlF₃ or MgF₃⁻). The optimized geometry for each structure has been found starting from experimental coordinates of one of them (PDBID: 1WQ1). Direct comparison of the experimental and computed geometry configurations in the immediate vicinity of the active site suggests that MgF₃⁻ is the most likely candidate for the phosphate analog in the experimental structure.     

Studies on propylamine transfer activity in anti-AdoDATO antibodies

Summary Based on a structural similarity to the transition state of a propylamine transfer reaction involved in polyamine biosynthesis, Sadenosyl-(5)-1,8-diamino-3-thiooctane (AdoDATO), the most potent inhibitor of spermidine synthases, was used as a hapten for mice immunization. From immunized mice sera, the IgG fractions were purified by means of affinity (protein A/G) chromatography. Sera and purified polyclonal antibodies from several mice were found to exert spermidine synthase-like activity. Moreover, by means means of hybridoma technology, 19 anti-AdoDATO hybridoma clones have been screened for propylamine transfer activity and at least 6 were found to produce catalytic antibodies. These findings indicate the presence in the sera of active spermidine synthase-like catalytic antibodies. The reported results for the first time evidence the feasibility of preparation of N-alkylating antibodies, widening the biotechnological perspectives of antibodies as biocatalysts.Abbreviations AdoDATO S-adenosyl-(5)-1,8-diaminothiooctane - TSA Transition state analog - decAdoMet S-adenosyl-(5)-3-methylthiopropylamine (decarboxylated adenosylmethionine) - KLH keyhole limpet hemocyanin - NMR nuclear magnetic resonance - BSA bovine serum albumine - WSC 1-ethyl-3-(dimethylaminopropyl)carbodiimide (water-soluble carbodiimide) - PBS phosphate buffer saline - OPD orthodiphenylenediamine - TCA trichloroacetic acid - SDS-PAGE sodium duodecylsulphate-polyacrylamide gel electrophoresis - SN2 bimolecular nucleophilic substitution; abzyme catalytic antibody - IgG immunoglobulin G     

Catalytic mechanism and energy barriers for butyrylcholinesterase-catalyzed hydrolysis of cocaine

The geometries of the transition states, intermediates, and prereactive enzyme-substrate complex and the corresponding energy barriers have been determined by performing hybrid quantum mechanical/molecular mechanical (QM/MM) calculations on butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)- and (+)-cocaine. The energy barriers were evaluated by performing QM/MM calculations with the QM method at the MP2/6-31+G* level and the MM method using the AMBER force field. These calculations allow us to account for the protein environmental effects on the transition states and energy barriers of these enzymatic reactions, showing remarkable effects of the protein environment on intermolecular hydrogen bonding (with an oxyanion hole), which is crucial for the transition state stabilization and, therefore, on the energy barriers. The calculated energy barriers are consistent with available experimental kinetic data. The highest barrier calculated for BChE-catalyzed hydrolysis of (-)- and (+)-cocaine is associated with the third reaction step, but the energy barrier calculated for the first step is close to the highest and is so sensitive to the protein environment that the first reaction step can be rate determining for (-)-cocaine hydrolysis catalyzed by a BChE mutant. The computational results provide valuable insights into future design of BChE mutants with a higher catalytic activity for (-)-cocaine.     

Modeling evolution of hydrogen bonding and stabilization of transition states in the process of cocaine hydrolysis catalyzed by human butyrylcholinesterase

Molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the prereactive enzyme-substrate complex, transition states, intermediates, and product involved in the process of human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)-cocaine. The computational results consistently reveal a unique role of the oxyanion hole (consisting of G116, G117, and A199) in BChE-catalyzed hydrolysis of cocaine, compared to acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine. During BChE-catalyzed hydrolysis of cocaine, only G117 has a hydrogen bond with the carbonyl oxygen (O31) of the cocaine benzoyl ester in the prereactive BChE-cocaine complex, and the NH groups of G117 and A199 are hydrogen-bonded with O31 of cocaine in all of the transition states and intermediates. Surprisingly, the NH hydrogen of G116 forms an unexpected hydrogen bond with the carboxyl group of E197 side chain and, therefore, is not available to form a hydrogen bond with O31 of cocaine in the acylation. The NH hydrogen of G116 is only partially available to form a weak hydrogen bond with O31 of cocaine in some structures involved in the deacylation. The change of the estimated hydrogen-bonding energy between the oxyanion hole and O31 of cocaine during the reaction process demonstrates how the protein environment can affect the energy barrier for each step of the BChE-catalyzed hydrolysis of cocaine. These insights concerning the effects of the oxyanion hole on the energy barriers provide valuable clues on how to rationally design BChE mutants with a higher catalytic activity for the hydrolysis of (-)-cocaine.     

Free-Energy Perturbation Simulation on Transition States and Redesign of Butyrylcholinesterase

It is recognized that an ideal anti-cocaine treatment is to accelerate cocaine metabolism by producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., butyrylcholinesterase (BChE)-catalyzed hydrolysis of cocaine. BChE mutants with a higher catalytic activity against (-)-cocaine are highly desired for use as an exogenous enzyme in humans. To develop a rational design for high-activity mutants, we carried out free-energy perturbation (FEP) simulations on various mutations of the transition-state structures in addition to the corresponding free-enzyme structures by using an extended FEP procedure. The FEP simulations on the mutations of both the free-enzyme and transition-state structures allowed us to calculate the mutation-caused shift of the free-energy change from the free enzyme (BChE) to the transition state, and thus to theoretically predict the mutation-caused shift of the catalytic efficiency (k_cat/K_M). The computational predictions are supported by the kinetic data obtained from the wet experiments, demonstrating that the FEP-based computational design approach is promising for rational design of high-activity mutants of an enzyme. One of the BChE mutants designed and discovered in this study has an ∼1800-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. The high-activity mutant may be therapeutically valuable.     

Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator

The mechanism of acid and enzymatic hydrolysis of the N-glycosidic bond of AMP has been investigated by fitting experimentally observed kinetic isotope effects [Parkin, D. W., & Schramm, V. L. (1987) Biochemistry (preceding paper in this issue)] to calculated kinetic isotope effects for proposed transition-state structures. The sensitivity of the transition-state calculations was tested by "arying the transition-state structure and comparing changes in the calculated kinetic isotope effects with the experimental values of the isotope effect measurements. The kinetic isotope effects for the acid-catalyzed hydrolysis of AMP are best explained by a transition state with considerable oxycarbonium character in the ribose ring, significant bonding remaining to the departing adenine ring, participation of a water nucleophile, and protonation of the adenine ring. A transition-state structure without preassociation of the water nucleophile cannot be eliminated by the data. Enzymatic hydrolysis of the N-glycosidic bond of AMP by AMP nucleosidase from Azotobacter vinelandii was analyzed in the absence and presence of MgATP, the allosteric activator that increases Vmax approximately 200-fold. The transition states for enzyme-catalyzed hydrolysis that best explain the kinetic isotope effects involve early SN1 transition states with significant bond order in the glycosidic bond and protonation of the adenine base. The enzyme enforces participation of an enzyme-bound water molecule, which has weak bonding to C1' in the transition state. Activation of AMP nucleosidase by MgATP causes the bond order of the glycosidic bond in the transition state to increase significantly. Hyperconjugation in the ribosyl group is altered by enzymatic stabilization of the oxycarbonium ion. This change is consistent with the interaction of an amino acid on the enzyme. Together, these changes stabilize a carboxonium-like transition-state complex that occurs earlier in the reaction pathway than in the absence of allosteric activator. In addition to the allosteric changes that alter transition-state structure, the presence of other inductive effects that are unobserved by kinetic isotope measurements is also likely to increase the catalytic rate.     

A Comparison of Experimental and Computational Methods for Mapping the Interactions Present in the Transition State for Folding of FKBP12

The folding pathway of FKBP12, a 107 residue / protein, has been characterised in detail using a combination of experimental and computational techniques. FKBP12 follows a two-state model of folding in which only the denatured and native states are significantly populated; no intermediate states are detected. The refolding rate constant in water is 4 s^-1 at 25 °C. Two different experimental strategies were employed for studying the transition state for folding. In the first case, a non-mutagenic approach was used and the unfolding and refolding of the wild-type protein measured as a function of experimental conditions such as temperature, denaturant, ligand and trifluoroethanol (TFE) concentration. These data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, whichis mainly formed by two long loops, is largely unstructured in the transition state. TFE experiments suggest that the -helix may be formed in the transition state. The second experimental approach involved using protein engineering techniques with -value analysis. Residue-specific information on the structure and energetics of the transition state can be obtained by this method. 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state, instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domainfrom -spectrin. For FKBP12, the central three strands of the -sheet (2, 4 and 5), comprise the most structured region of the transition state. In particular Val 101, which is one of the most highly buried residues and located in the middle of the central -strand,makes approximately 60% of its native interactions. The outer -strands, and the ends of the central -strands are formed to a lesser degree. The short -helix is largely unstructured in the transition state as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side chains within -strands 2, 4 and 5 and the C-terminus of the -helix. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. High-temperature molecular dynamic simulations on the unfoldingpathway of FKBP12 are in good agreement with the experimental results.     

Catalytic mechanism of the inverting N-acetylglucosaminyltransferase I: DFT quantum mechanical model of the reaction pathway and determination of the transition state structure

The complex N-glycan structures on glycoproteins play important roles in cell adhesion and recognition events in metazoan organisms. A critical step in the biosynthetic pathway leading from high mannose to these complex structures includes the transfer of N-acetylglucosamine (GlcNAc) to a mannose residue by the inverting N-acetylglucosaminyltransferase I (GnT-I). The catalytic mechanism of this enzymatic reaction is explored herein using DFT quantum chemical methods. The computational model used to follow the reaction is based on the X-ray crystallographic structure of GnT-I and contains 127 atoms that represent fragments of residues critical for the substrate binding and catalysis. The mechanism of the catalytic reaction was monitored by means of a 2D potential energy map calculated as a function of predefined reaction coordinates at the B3LYP/6-31G** level. This potential energy surface revealed one transition state associated with a reaction pathway following a concerted mechanism. The reaction barrier was estimated, and the structure of the transition state was characterized at the B3LYP/6-311++G**// B3LYP/6-31G** level.     

An investigation of the pyranose ring interconversion path of alpha-L-idose calculated using density functional theory

The interconversion pathways of the pyranose ring conformation of alpha-L-idose from a (4)C1 chair to other conformations were investigated using density functional calculations. From these calculations, four different ring interconversion paths and their transition state structures from the (4)C1 chair to other conformations, such as B(3,O), and (1)S3, were obtained. These four transition-state conformations cover four possible combinations of the network patterns of the hydroxyl group hydrogen bonds (clockwise and counterclockwise) and the conformations of the primary alcohol group (tg and gg). The optimized conformations, transition states, and their intrinsic reaction coordinates (IRC) were all calculated at the B3LYP/6-31G** level. The energy differences among the structures obtained were evaluated at the B3LYP/6-311++G** level. The optimized conformations indicate that the conformers of (4)C1, (2)S(O), and B(3,O) have similar energies, while (1)S3 has a higher energy than the others. The comparison of the four transition states and their ring interconversion paths, which were confirmed using the IRC calculation, suggests that the most plausible ring interconversion of the alpha-L-idopyranose ring occurs between (4)C1 and B(3,O) through the E3 envelope, which involves a 5.21 kcal/mol energy barrier.     

Virtual screening of mandelate racemase mutants with enhanced activity based on binding energy in the transition state

Mandelate racemase (MR) is a promising candidate for the dynamic kinetic resolution of racemates. However, the poor activity of MR towards most of its non-natural substrates limits its widespread application. In this work, a virtual screening method based on the binding energy in the transition state was established to assist in the screening of MR mutants with enhanced catalytic efficiency. Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening. The number of mutants for experimental validation was brought down to 17 by the virtual screening method, among which 14 variants turned out to possess improved catalytic efficiency. The variant V26I/Y54V showed 5.2-fold higher catalytic efficiency (k_cat/K_m) towards R-3-chloromandelic acid than that observed for the wild-type enzyme. Using this strategy, mutants were successfully obtained for two other substrates, R-mandelamide and R-2-naphthylglycolate (V26I and V29L, respectively), both with a 2-fold improvement in catalytic efficiency. These results demonstrated that this method could effectively predict the trend of mutational effects on catalysis. Analysis from the energetic and structural assays indicated that the enhanced interactions between the active sites and the substrate in the transition state led to improved catalytic efficiency. It was concluded that this virtual screening method based on the binding energy in the transition state was beneficial in enzyme rational redesign and helped to better understand the catalytic properties of the enzyme.     

Binding of a Transition State Analog to Newly Synthesized Rubisco

Radioactive amino acids, when added to isolated pea chloroplasts or chloroplast extracts engaged in protein synthesis, are incorporated into Rubisco large subunits that co-migrate with native Rubisco during nondenaturing electrophoresis. We have added the transition state analog 2′-carboxyarabinitol bisphosphate (CABP) to chloroplast extracts after in organello or in vitro incorporation of radioactive amino acids into Rubisco large subunits. Upon addition of CABP the radioactive bands co-migrating with native Rubisco undergo a readily detected shift in electrophoretic mobility just as the native enzyme, thus demonstrating the ability of the newly assembled molecules to interact with this transition state analog.     

Transition state analysis of the complex between coagulation factor VIIa and tissue factor: suggesting a sequential domain-binding pathway

Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Phi values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules.     

Computational studies on nonenzymatic and enzymatic pyridoxal phosphate catalyzed decarboxylations of 2-aminoisobutyrate

A computational study of nonenzymatic and enzymatic pyridoxal phosphate-catalyzed decarboxylation of 2-aminoisobutyrate (AIB) is presented. Four prototropic isomers of a model aldimine between AIB and 5'-deoxypyridoxal, with acetate interacting with the pyridine nitrogen, were employed in calculations of both gas phase and water model (PM3 and PM3-SM3) decarboxylation reaction paths. Calculations employing the transition state structures obtained for the four isomers allow the demonstration of stereoelectronic effects in transition state stabilization as well as a separation of the contributions of the Schiff base and pyridine ring moieties to this stabilization. The unprotonated Schiff base contribution (approximately 16 kcal/mol) is larger than that of the pyridine ring even when it is protonated (approximately 10 kcal/mol), providing an explanation of the catalytic power of pyruvoyl-dependent amino acid decarboxylases. An active site model of dialkylglycine decarboxylase was constructed and validated, and enzymatic decarboxylation reaction paths were calculated. The reaction coordinate is shown to be complex, with proton transfer from Lys272 to the coenzyme C4' likely simultaneous with C alpha--CO(2)(-) bond cleavage. The proposed concerted decarboxylation/proton-transfer mechanism provides a simple explanation for the observed specificity of this enzyme toward oxidative decarboxylation.     

Diverse structural solutions to catalysis in a family of antibodies

BACKGROUND: Small organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation. RESULTS: The structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ. CONCLUSIONS: Our results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.     

How similar are enzyme active site geometries derived from quantum mechanical theozymes to crystal structures of enzyme‐inhibitor complexes? Implications for enzyme design

Quantum mechanical optimizations of theoretical enzymes (theozymes), which are predicted catalytic arrays of biological functionalities stabilizing a transition state, have been carried out for a set of nine diverse enzyme active sites. For each enzyme, the theozyme for the rate-determining transition state plus the catalytic groups modeled by side-chain mimics was optimized using B3LYP/6-31G(d) or, in one case, HF/3-21G(d) quantum mechanical calculations. To determine if the theozyme can reproduce the natural evolutionary catalytic geometry, the positions of optimized catalytic atoms, i.e., covalent, partial covalent, or stabilizing interactions with transition state atoms, are compared to the positions of the atoms in the X-ray crystal structure with a bound inhibitor. These structure comparisons are contrasted to computed substrate-active site structures surrounded by the same theozyme residues. The theozyme/transition structure is shown to predict geometries of active sites with an average RMSD of 0.64 A from the crystal structure, while the RMSD for the bound intermediate complexes are significantly higher at 1.42 A. The implications for computational enzyme design are discussed.     

Mechanism of ATP turnover inhibition in the EJC

The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF₃, the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC–ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5–RNA–ADP–AlF₃, suggesting that the use of ADP–AlF₃ may be a valuable tool for examining DEAD-box ATPases in general.