Induced-fit Mechanism Research Articles

The pseudotorsional space of RNA.

The characterization of the conformational landscape of the RNA backbone is rather complex due to the ability of RNA to assume a large variety of conformations. These backbone conformations can be depicted by pseudotorsional angles linking RNA backbone atoms, from which Ramachandran-like plots can be built. We explore here different definitions of these pseudotorsional angles, finding that the most accurate ones are the traditional η (eta) and θ (theta) angles, which represent the relative position of RNA backbone atoms P and C4'. We explore the distribution of η - θ in known experimental structures, comparing the pseudotorsional space generated with structures determined exclusively by one experimental technique. We found that the complete picture only appears when combining data from different sources. The maps provide a quite comprehensive representation of the RNA accessible space, which can be used in RNA-structural predictions. Finally, our results highlight that protein interactions lead to significant changes in the population of the η - θ space, pointing toward the role of induced-fit mechanisms in protein-RNA recognition.

Conformational Selection and Induced Fit: The Behavior of Two Homologous Proteases

AbstractProtein‐ligand interactions are crucial for many cellular processes, with details of the binding mechanism being discussed as essential for biological functions. Interestingly, protein binding often involves conformational changes between two or more states, whereby different binding mechanisms are possible even with a simple two‐state description. Two models are widely used to portray protein‐ligand interactions: Induced fit and conformational selection. However, distinguishing them experimentally is challenging. Single‐molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool to resolve structural dynamics at the level of single proteins. Here, we investigated immobilized Zika virus (ZIKV) and dengue virus (DENV2) NS2B‐NS3 proteases using smFRET to compare their conformational changes upon binding to competitive small molecule inhibitors. The analysis of the smFRET data allowed us to distinguish between induced fit and conformational selection models and assign the binding mechanism from the kinetic parameters obtained. Although DENV and ZIKV protease are proteins with high structural similarities, our results reveal that they have opposite binding mechanisms for competitive ligands. While the protein‐ligand interaction in the ZIKV protease follows an induced fit mechanism, the DENV protease follows the conformational selection mechanism.

Adaptive Pore Opening to Form Tailored Adsorption Sites in a Cooperatively Flexible Framework Enables Record Inverse Propane/Propylene Separation.

A proposed low-energy alternative to the separation of alkanes from alkenes by energy-intensive cryogenic distillation is separation by porous adsorbents. Unfortunately, most adsorbents preferentially take up the desired, high-value major component alkene, requiring frequent regeneration. Adsorbents with inverse selectivity for the minor component alkane would enable the direct production of purified, reagent-grade alkene, greatly reducing global energy consumption. However, such materials are exceedingly rare, especially for propane/propylene separation. Here, we report that through adaptive and spontaneous pore size and shape adaptation to optimize an ensemble of weak noncovalent interactions, the structurally responsive metal-organic framework CdIF-13 (sod-Cd(benzimidazolate)2) exhibits inverse selectivity for propane over propylene with record-setting separation performance under industrially relevant temperature, pressure, and mixture conditions. Powder synchrotron X-ray diffraction measurements combined with first-principles calculations yield atomic-scale insight and reveal the induced fit mechanism of adsorbate-specific pore adaptation and ensemble interactions between ligands and adsorbates. Dynamic column breakthrough measurements confirm that CdIF-13 displays selectivity under mixed-component conditions of varying ratios, with a record measured selectivity factor of α ≈ 3 at 95:5 propylene:propane at 298 K and 1 bar. When sequenced with a low-cost rigid adsorbent, we demonstrated the direct purification of propylene under ambient conditions. This combined atomic-level structural characterization and performance testing firmly establishes how cooperatively flexible materials can be capable of unprecedented separation factors.

Host Membranes as Drivers of Virus Evolution.

The molecular mechanisms controlling the adaptation of viruses to host cells are generally poorly documented. An essential issue to resolve is whether host membranes, and especially lipid rafts, which are usually considered passive gateways for many enveloped viruses, also encode informational guidelines that could determine virus evolution. Due to their enrichment in gangliosides which confer an electronegative surface potential, lipid rafts impose a first control level favoring the selection of viruses with enhanced cationic areas, as illustrated by SARS-CoV-2 variants. Ganglioside clusters attract viral particles in a dynamic electrostatic funnel, the more cationic viruses of a viral population winning the race. However, electrostatic forces account for only a small part of the energy of raft-virus interaction, which depends mainly on the ability of viruses to form a network of hydrogen bonds with raft gangliosides. This fine tuning of virus-ganglioside interactions, which is essential to stabilize the virus on the host membrane, generates a second level of selection pressure driven by a typical induced-fit mechanism. Gangliosides play an active role in this process, wrapping around the virus spikes through a dynamic quicksand-like mechanism. Viruses are thus in an endless race for access to lipid rafts, and they are bound to evolve perpetually, combining speed (electrostatic potential) and precision (fine tuning of amino acids) under the selective pressure of the immune system. Deciphering the host membrane guidelines controlling virus evolution mechanisms may open new avenues for the design of innovative antivirals.

Insight into the Binding of First- and Second-Generation PET Tracers to 4R and 3R/4R Tau Protofibrils.

Primary supranuclear palsy (PSP) is a rare neurodegenerative disease that perturbs body movement, eye movement, and walking balance. Similar to Alzheimer's disease (AD), the abnormal aggregation of tau fibrils in the central neuronal and glial cells is a major hallmark of PSP disease. In this study, we use multiple approaches, including docking, molecular dynamics, and metadynamics simulations, to investigate the binding mechanism of 10 first- and second-generations of PET tracers for PSP tau and compare their binding in cortical basal degeneration (CBD) and AD tauopathies. Structure-activity relationships, binding preferences, the nature of ligand binding in terms of basic intermolecular interactions, the role of polar/charged residues, induced-fit mechanisms, grove closures, and folding patterns for the binding of these tracers in PSP, CBD, and AD tau fibrils are evaluated and discussed in detail in order to build a holistic picture of what is essential for the binding and also to rank the potency of the different tracers. For example, we found that the same tracer shows different binding preferences for the surface sites of tau fibrils that are intrinsically distinct in the folding patterns. Results from the metadynamics simulations predict that PMPBB3 and PBB3 exhibit the strongest binding free energies onto the Q276[I277]I278, Q351[S352]K353, and N368[K369]K370 sites of PSP than the other explored tracers, indicating a solid preference for vdW and cation-π interactions. Our results also reproduced known preferences of tracers, namely, that MK6240 binds better to AD tau than CBD tau and PSP tau and that CBD2115, PI2620, and PMPBB3 are 4R tau binders. These findings fill in the well-sought-after knowledge gap in terms of these tracers' potential binding mechanisms and will be important for the design of highly selective novel PET tracers for tauopathies.

Utilization of Cofactor Binding Energy for Enzyme Catalysis: Formate Dehydrogenase-Catalyzed Reactions of the Whole NAD Cofactor and Cofactor Pieces.

The pressure to optimize enzymatic rate accelerations has driven the evolution of the induced-fit mechanism for enzyme catalysts where the binding interactions of nonreacting phosphodianion or adenosyl substrate pieces drive enzyme conformational changes to form protein substrate cages that are activated for catalysis. We report the results of experiments to test the hypothesis that utilization of the binding energy of the adenosine 5'-diphosphate ribose (ADP-ribose) fragment of the NAD cofactor to drive a protein conformational change activates Candida boidinii formate dehydrogenase (CbFDH) for catalysis of hydride transfer from formate to NAD+. The ADP-ribose fragment provides a >14 kcal/mol stabilization of the transition state for CbFDH-catalyzed hydride transfer from formate to NAD+. This is larger than the ca. 6 kcal/mol stabilization of the ground-state Michaelis complex between CbFDH and NAD+ (KNAD = 0.032 mM). The ADP, AMP, and ribose 5'-phosphate fragments of NAD+ activate CbFDH for catalysis of hydride transfer from formate to nicotinamide riboside (NR). At a 1.0 M standard state, these activators stabilize the hydride transfer transition states by ≈5.5 (ADP), 5.5 (AMP), and 4.4 (ribose 5'-phosphate) kcal/mol. We propose that activation by these cofactor fragments is partly or entirely due to the ion-pair interaction between the guanidino side chain cation of R174 and the activator phosphate anion. This substitutes for the interaction between the α-adenosyl pyrophosphate anion of the whole NAD+ cofactor that holds CbFDH in the catalytically active closed conformation.

Mode of substrate binding for ketohexokinase across isozymes and species implies an induced-fit mechanism

Mode of substrate binding for ketohexokinase across isozymes and species implies an induced-fit mechanism

Low molecular weight ligands bind to CYP3A4 via a branched induced fit mechanism: Implications for O2 binding.

Low molecular weight ligands bind to CYP3A4 via a branched induced fit mechanism: Implications for O2 binding.

Intermediate-aided allostery mechanism for α-glucosidase by Xanthene-11v as an inhibitor using residue interaction network analysis

Exploring allosteric inhibition and the discovery of new inhibitor binding sites are important studies in protein regulation mechanisms and drug discovery. Structural and network-based analyses of trajectories resulting from molecular dynamics (MD) simulations have been developed to discover protein dynamics, landscape, functions, and allosteric regions. Here, an experimentally suggested non-competitive inhibitor, xanthene-11v, was considered to explore its allosteric inhibition mechanism in α-glucosidase MAL12. Comparative structural and network analyses were applied to eight 250 ns independent MD simulations, four of which were performed in the free state and four of which were performed in ligand-bound forms. Projected two-dimensional free energy landscapes (FEL) were constructed from the probabilistic distribution of conformations along the first two principal components. The post-simulation analyses of the coordinates, side-chain torsion angles, non-covalent interaction networks, network communities, and their centralities were performed on α-glucosidase conformations and the intermediate sub-states. Important communities of residues have been found that connect the allosteric site to the active site. Some of these residues like Thr307, Arg312, TYR344, ILE345, Phe357, Asp406, Val407, Asp408, and Leu436 are the key messengers in the transition pathway between allosteric and active sites. Evaluating the probability distribution of distances between gate residues including Val407 in one community and Phe158, and Pro65 in another community depicted the closure of this gate due to the inhibitor binding. Six macro states of protein were deduced from the topology of FEL and analysis of conformational preference of free and ligand-bound systems to these macro states shows a combination of lock-and-key, conformational selection, and induced fit mechanisms are effective in ligand binding. All these results reveal structural states, allosteric mechanisms, and key players in the inhibition pathway of α-glucosidase by xanthene-11v.

Mutual induced-fit mechanism drives binding between intrinsically disordered Bim and cryptic binding site of Bcl-xL

The intrinsically disordered region (IDR) of Bim binds to the flexible cryptic site of Bcl-xL, a pro-survival protein involved in cancer progression that plays an important role in initiating apoptosis. However, their binding mechanism has not yet been elucidated. We have applied our dynamic docking protocol, which correctly reproduced both the IDR properties of Bim and the native bound configuration, as well as suggesting other stable/meta-stable binding configurations and revealed the binding pathway. Although the cryptic site of Bcl-xL is predominantly in a closed conformation, initial binding of Bim in an encounter configuration leads to mutual induced-fit binding, where both molecules adapt to each other; Bcl-xL transitions to an open state as Bim folds from a disordered to an α-helical conformation while the two molecules bind each other. Finally, our data provides new avenues to develop novel drugs by targeting newly discovered stable conformations of Bcl-xL.

Synthesis of theories on cellular powering, coherence, homeostasis and electro-mechanics: Murburn concept and evolutionary perspectives.

If evolution was/is a fact, a simplified/unifying approach to explain cellular physiology is warranted. Such a perspective should agree with the thermodynamic, kinetic, structural, and operational-probabilistic considerations; without invoking overt intelligence or determinism, and must enable a synthesis from chaos. In this regard, we first list salient theories in cellular physiology for (i) powering (generation of chemical/heat energy), (ii) coherence (interconnectivity and workability as a unit), (iii) homeostasis (metabolizing and expelling of unfamiliar/unwanted materials, maintaining concentration/volume), and (iv) cellular electrical-mechanical activities. While doing so, we discuss the scopes and limitations of (a) the classical active-site affinity and recognition-based modality of lock-key and induced-fit enzyme-catalytic mechanisms established by Fischer/Koshland, (b) membrane-pump hypothesis acclaimed by biologists-physicians and historically championed by the British Nobel-laureates like Hodgkin-Huxley-Katz-Mitchell, and (c) association-induction hypothesis advocated by physicists-physiologists from various parts of the world, for example, Gilbert Ling (China-USA), Gerald Pollack (USA), Ludwig Edelmann (Germany), Vladimir Matveev (Russia), and so on. We apply murburn concept (from "mured burning," capturing the thesis that one-electron redox equilibriums involving diffusible reactive species play vital roles in maintaining life order) to amalgamate several core cellular functions and further discuss the prospects for establishing the continuum of the principles of physics in biology.

SARS-CoV-2 envelope protein attain Kac mediated dynamical interaction network to adopt ‘histone mimic’ at BRD4 interface

Interface mimicry, achieved by recognition of host-pathogen interactions, is the basis by which pathogen proteins can hijack the host machinery. The envelope (E) protein of SARS-CoV-2 is reported to mimic the histones at the BRD4 surface via establishing the structural mimicry; however, the underlying mechanism of E protein mimicking the histones is still elusive. To explore the mimics at dynamic and structural residual network level an extensive docking, and MD simulations were carried out in a comparative manner between complexes of H3-, H4-, E-, and apo-BRD4. We identified that E peptide is able to attain an ‘interaction network mimicry’, as its acetylated lysine (Kac) achieves orientation and residual fingerprint similar to histones, including water-mediated interactions for both the Kac positions. We identified Y59 of E, playing an anchor role to escort lysine positioning inside the binding site. Furthermore, the binding site analysis confirms that E peptide needs a higher volume, similar to the H4-BRD4 where both the lysine’s (Kac5 and Kac8) can accommodate nicely, however, the position of Kac8 is mimicked by two additional water molecules other than four water-mediated bridging’s, strengthening the possibility that E peptide could hijack host BRD4 surface. These molecular insights seem pivotal for mechanistic understanding and BRD4-specific therapeutic intervention. KEY POINTS Molecular mimicry is reported in hijacking and then outcompeting the host counterparts so that pathogens can rewire their cellular function by overcoming the host defense mechanism. The molecular recognition process is the basis of molecular mimicry. The E peptide of SARS-CoV-2 is reported to mimic host histone at the BRD4 surface by utilizing its C-terminally placed acetylated lysine (Kac63) to mimic the N-terminally placed acetylated lysine Kac5GGKac8 histone (H4) by interaction network mimicry identified through microsecond molecular dynamics (MD) simulations and post-processing extensive analysis. There are two steps to mimic: firstly, tyrosine residues help E to anchor at the BRD4 surface to position Kac and increase the volume of the pocket. Secondary, after positioning of Kac, a common durable interaction network N140:Kac5; Kac5:W1; W1:Y97; W1:W2; W2:W3; W3:W4; W4:P82 is established between Kac5, with key residues P82, Y97, N140, and four water molecules through water mediate bridge. Furthermore, the second acetylated lysine Kac8 position and its interaction as polar contact with Kac5 were also mimicked by E peptide through interaction network P82:W5; W5:Kac63; W5:W6; W6:Kac63. The binding event at BRD4/BD1 seems an induced-fit mechanism as a bigger binding site volume was identified at H4-BRD4 on which E peptide attains its better stability than H3-BRD4. We identified the tyrosine residue Y59 of E that acts like an anchor on the BRD4 surface to position Kac inside the pocket and attain the interaction network by using aromatic residues of the BRD4 surface. Communicated by Ramaswamy H. Sarma

Structural Complementarity of Bruton’s Tyrosine Kinase and Its Inhibitors for Implication in B-Cell Malignancies and Autoimmune Diseases

Bruton's tyrosine kinase (BTK) is a critical component in B-cell receptor (BCR) signaling and is also expressed in haematogenic and innate immune cells. Inhibition of BTK hyperactivity is implicated in B-cell malignancies and autoimmune diseases. This review derives the structural complementarity of the BTK-kinase domain and its inhibitors from recent three-dimensional structures of inhibitor-bound BTK in the protein data bank (PDB). Additionally, this review analyzes BTK-mediated effector responses of B-cell development and antibody production. Covalent inhibitors contain an α, β-unsaturated carbonyl moiety that forms a covalent bond with Cys481, stabilizing αC-helix in inactive-out conformation which inhibits Tyr551 autophosphorylation. Asn484, located two carbons far from Cys481, influences the stability of the BTK-transition complex. Non-covalent inhibitors engage the BTK-kinase domain through an induced-fit mechanism independent of Cys481 interaction and bind to Tyr551 in the activation kink resulting in H3 cleft, determining BTK selectivity. Covalent and non-covalent binding to the kinase domain of BTK shall induce conformational changes in other domains; therefore, investigating the whole-length BTK conformation is necessary to comprehend BTK's autophosphorylation inhibition. Knowledge about the structural complementarity of BTK and its inhibitors supports the optimization of existing drugs and the discovery of drugs for implication in B-cell malignancies and autoimmune diseases.

Anionic phospholipids control mechanisms of GPCR-G protein recognition

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

Structural basis of GABA reuptake inhibition.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1), a member of the neurotransmitter sodium symporter (NSS) family, regulates neuronal excitation by clearing the synaptic cleft of the main inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating GABA levels in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we present the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor, the anticonvulsant tiagabine. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine. Tiagabine's pharmacodynamic profile, confirmed by our experimental data, suggests initial binding to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step induced-fit mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators.

Molecular mechanism of binding between a therapeutic RNA aptamer and its protein target VEGF: A molecular dynamics study.

Macugen is a therapeutic RNA aptamer against vascular endothelial growth factor (VEGF)-165, the VEGF isoform primarily responsible for angiogenesis. It has been reported that Macugen inhibits angiogenesis by specifically binding to the heparin binding domain (HBD) of VEGF165. The mechanism of the molecular recognition between HBD and Macugen is investigated here using all-atom molecular dynamics simulations. We find that Macugen recognizes HBD by an induced-fit mechanism with major conformational changes in Macugen and almost no changes in the structure of HBD, whereas HBD recognizes Macugen by a conformational selection mechanism.

W254 in furin functions as a molecular gate promoting anti-viral drug binding: Elucidation of putative drug tunneling and docking by non-equilibrium molecular dynamics

Furins are serine endoproteases that process precursor proteins into their biologically active forms, and they play essential roles in normal metabolism and disease presentation, including promoting expression of bacterial virulence factors and viral pathogenesis. Thus, furins represent vital targets for development of antimicrobial and antiviral therapeutics. Recent experimental evidence indicated that dichlorophenyl (DCP)-pyridine “BOS” drugs (e.g., BOS-318) competitively inhibit human furin by an induced-fit mechanism in which tryptophan W254 in the furin catalytic cleft (FCC) functions as a molecular gate, rotating nearly 180o through a steep energy barrier about its chi-1 dihedral to an “open” orientation, exposing a buried (i.e., cryptic) hydrophobic pocket 1. Once exposed, the non-polar DCP group of BOS-318, and similar halo-phenyl groups of analogs, enter the cryptic pocket, stabilizing drug binding. Here, we demonstrate flexible-receptor docking of BOS-318 (and various analogs) was unable to emulate the induced-fit motif, even when tryptophan was replaced with less bulky phenylalanine or glycine. While either substitution allowed access to the hydrophobic pocket for most ligands tested, optimal binding was observed only for W254, inferring a stabilizing effect of the indole sidechain. Furthermore, non-equilibrium steered molecular dynamics (sMD) in which the bound drugs (or their fragments) were extracted from the FCC did not cause closure of the open W254 gate, consistent with the thermodynamic stability of the open or closed W254 orientations. Finally, interactive molecular dynamics (iMD) revealed two putative conduits of drug entry and binding into the FCC, each coupled with W254 dihedral rotation and opening of the cryptic pocket. The iMD simulations further revealed ligand entry and binding in the FCC is likely driven in part by energy fluxes stemming from disruption and re-formation of ligand and protein solvation shells during drug migration from the solution phase into the FCC.

Stretching the chains: the destabilizing impact of Cu2+ and Zn2+ ions on K48-linked diubiquitin.

Ubiquitin signalling and metal homeostasis play key roles in controlling several physiological cellular activities, including protein trafficking and degradation. While some relationships between these two biochemical pathways have started to surface, our knowledge of their interplay remains limited. Here, we employ a variety of techniques, such as circular dichroism, differential scanning calorimetry, pressure perturbation calorimetry, fluorescence emission, SDS-PAGE, and small-angle X-ray scattering (SAXS) to evaluate the impact of Cu2+ and Zn2+ ions on the structure and stability of K48 linked diubiquitin (K48-Ub2), a simple model for polyubiquitin chains. The SAXS analysis results show that the structure of the metal-free protein is similar to that observed when the protein is bound to the E2 conjugating enzyme, lending support to the idea that the structure of unanchored K48-linked ubiquitin chains is sufficient for identification by conjugating enzymes without the need for an induced fit mechanism. Our results indicate that K48-Ub2 can coordinate up to four metal ions with both copper and zinc ions inducing slight changes to the secondary structure of the protein. However, we noted significant distinctions in their impacts on protein stability and overall architecture. Specifically, Cu2+ ions resulted in a destabilization of the protein structure, which facilitated the formation of dimer aggregates. Next, we observed a shift in the conformational dynamics of K48-Ub2 toward less compact and more flexible states upon metal ion binding, with Zn2+ inducing a more significant effect than Cu2+ ions. Our structural modelling study demonstrates that both metal ions induced perturbations in the K48-Ub2 structure, leading to the separation of the two monomers thus inhibiting interactions with E2 enzymes. In conclusion, the findings from this study enhance our comprehension of the mechanisms underlying Ub chains recognition. Moreover, they strengthen the notion that drug discovery initiatives aimed at targeting metal-mediated disruptions in Ub signaling hold great potential for treating a wide range of diseases that stem from abnormal protein accumulation.

Discovery, Structure, and Mechanism of a Class II Sesquiterpene Cyclase.

Terpene cyclases (TCs), extraordinary enzymes that create the structural diversity seen in terpene natural products, are traditionally divided into two classes, class I and class II. Although the structural and mechanistic features of class I TCs are well-known, the corresponding details in class II counterparts have not been fully characterized. Here, we report the genome mining discovery and structural characterization of two class II sesquiterpene cyclases (STCs) from Streptomyces. These drimenyl diphosphate synthases (DMSs) are the first STCs shown to possess β,γ-didomain architecture. High-resolution X-ray crystal structures of DMS from Streptomyces showdoensis (SsDMS) in complex with both a farnesyl diphosphate and Mg2+ unveiled an induced-fit mechanism, with an unprecedented Mg2+ binding mode, finally solving one of the lingering questions in class II TC enzymology. This study supports continued genome mining for novel bacterial TCs and provides new mechanistic insights into canonical class II TCs that will lead to advances in TC engineering and synthetic biology.

Adenylate Kinase-Catalyzed Reactions of AMP in Pieces: Specificity for Catalysis at the Nucleoside Activator and Dianion Catalytic Sites.

The pressure to optimize the enzymatic rate acceleration for adenylate kinase (AK)-catalyzed phosphoryl transfer has led to the evolution of an induced-fit mechanism, where the binding energy from interactions between the protein and substrate adenosyl group is utilized to drive a protein conformational change that activates the enzyme for catalysis. The adenine group of adenosine contributes 11.8 kcal mol-1 to the total ≥14.7 kcal mol-1 adenosine stabilization of the transition state for AK-catalyzed phosphoryl transfer to AMP. The relative third-order rate constants for activation of adenylate kinase, by the C-5 truncated adenosine 1-(β-d-erythrofuranosyl)adenine (EA), for catalysis of phosphoryl transfer from ATP to phosphite dianion (HP, kcat/KHPKAct = 260 M-2 s-1), fluorophosphate (47 M-2 s-1), and phosphate (9.6 M-2 s-1), show that substitution of -F for -H and of -OH for -H at HP results, respectively, in decreases in the reactivity of AK for catalysis of phosphoryl transfer due to polar and steric effects of the -F and -OH substituents. The addition of a 5'-CH2OH to the EA activator results in a 3.0 kcal mol-1 destabilization of the transition state for AK-activated phosphoryl transfer to HP due to a steric effect. This is smaller than the 8.3 kcal mol-1 steric effect of the 5'-CH2OH substituent at OMP on HP-activated OMPDC-catalyzed decarboxylation of 1-(β-d-erythrofuranosyl)orotate. The 2'-OH ribosyl substituent shows significant interactions with the transition states for AK-catalyzed phosphoryl transfer from ATP to AMP and for adenosine-activated AK-catalyzed phosphoryl transfer from ATP to HP.