Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.
Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active site residues not only reduces the catalytic activity of these enzymes, but also dramatically affects substrate binding. Employing the cyclophilin A (CypA) PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the HIV-1 capsid (CA(N)) protein, we demonstrate here how to dissect residue specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of CypA active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based on a peptide assay only, catalyze the HIV capsid with wild-type activity, but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.
The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.
The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.
The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment–protein complex responsible for light‐driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton‐induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near‐identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid–base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH‐dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β‐barrel domain in response to the fluctuating acidity of the thylakoid lumen.
Identifying functional partners for protein/protein interactions can be a difficult challenge. We proposed the use of the operon structure of the Caenorhabditis elegans genome as a 'New Gene-Finding Tool' (Eichmueller et al, JBC, 2004, 279:7136) that could be functionally translated to the human system. Here we show the validity of this approach by studying the predicted functional interaction between ICln and HSPC038. In Caenorhabditis elegans, the gene encoding for the ICln homolog (icln-1) is embedded in an operon with two other genes, Nx (the human homolog of Nx is HSPC038) and Ny. ICln is a highly conserved, ubiquitously expressed multifunctional protein that plays a critical role in the regulatory volume decrease after cell swelling. Following hypotonic stress, ICln translocates from the cytosol to the plasma membrane, where it has been proposed to participate in the activation of the swelling induced chloride current (IClswell). Here we show that the interaction between human ICln and HSPC038 plays a role in volume regulation after cell swelling and that HSPC038 acts as an escort, directing ICln to the cell membrane after cell swelling and facilitating the activation of IClswell. Assessment of the NMR structure of HSPC038 showed the presence of a zinc finger motif. Moreover, NMR and additional biochemical techniques enabled us to identify the putative ICln/HSPC038 interacting sites, thereby explaining the functional interaction of both proteins on a molecular level.
Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme’s conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme’s function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK’s conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme’s conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme’s active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.
A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis1, 2, 3, 4, 5. The concept of conformational substates has been described6; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis–trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously3, 7, 8, 9, 10, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.
Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.
Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.
SummaryPhosphorylation is a common mechanism for activating proteins within signaling pathways. Yet, the molecular transitions between the inactive and active conformational states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of a signaling protein, nitrogen regulatory protein C (NtrC), by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding and show that only a rarely populated, pre-existing active conformation is energetically stabilized by phosphorylation. Using nuclear magnetic resonance (NMR) dynamics, we test an atomic scale pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009). The data show that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results unravel atomistic details of native-state protein energy landscapes by expanding the knowledge about ground states to transition landscapes.
Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.
The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate-enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent 'closed' state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia the switch to secretion of effector proteins is induced first after that intimate contact between the bacterium and its eukaryotic targetcell has been established and the T3SS proteins YscP and YscU are playing a central role in thisprocess. Here we identify the molecular details of the YscP binding site on YscU by means o fnuclear magnetic resonance (NMR) spectroscopy. The binding interface is centeredon the C-terminal domain of YscU. Disruptingthe YscU/YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the bindingof YscP to the slowly self-cleaving YscU variantP264A conferred significant protection againstauto-proteolysis. The YscP mediated inhibition of YscU auto-proteolysis suggest that the cleavage event may act as a timing switch in the regulationof early vs. late T3SS substrates. We also show that YscUC binds to the inner-rod protein YscI with a Kd of 3.8 μM and with one-to-one stoichiometry. The significant similarity between different members of the YscU, YscP, YscI families suggests that the protein-protein interactions discussed in this study are alsorelevant for other T3SS-containing Gram-negative bacteria.
Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson's disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on (15)N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain β-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.
Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-angstrom X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic side-chains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.
It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.
An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.
Acute myeloid leukemia 1 (AML1), also denoted Runx1, is a transcription factor essential for hematopoiesis, and the AML1 gene is the most common target of chromosomal translocations in human leukemias. AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. Here we show that both subunits of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1. The regulatory CN subunit binds AML1 with a higher affinity and in addition also interacts with the isolated runt homology domain. The related Runx2 transcription factor, which is essential for bone formation, also interacts with CN. A constitutively active derivative of CN is shown to activate synergistically the GM-CSF promoter/enhancer together with AML1 or Runx2. We also provide evidence that relief of the negative effect of the AML1 sites is important for Ca(2+) activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases this Ca(2+) activation. Both subunits of CN interact with AML1 in coimmunoprecipitation analyses, and confocal microscopy analysis of cells expressing fluorescence-tagged protein derivatives shows that CN can be recruited to the nucleus by AML1 in vivo. Mutant analysis of the GM-CSF promoter shows that the Ets1 binding site of the promoter is essential for the synergy between AML1 and CN in Jurkat T cells. Analysis of the effects of inhibitors of the protein kinase glycogen synthase kinase-3beta and in vitro phosphorylation/dephosphorylation analysis of Ets1 suggest that glycogen synthase kinase-3beta-phosphorylated Ets1 is a target of AML1-recruited CN phosphatase at the GM-CSF promoter.
Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as AFM, thioflavin-T fluorescence, circular dichroism and gas phase electrophoretic mobility macromolecule analysis we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pHs and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding the inflection point of the growth phase; (ii) an abrupt cut-off between growth and disaggregation at pH 8-10; (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time AFM and thioflavin-T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca. 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.
This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.
Understanding enzyme mechanisms is essential for unraveling the complex molecular machinery of life. In this review, we survey the field of computational enzymology, highlighting key principles governing enzyme mechanisms and discussing ongoing challenges and promising advances. Over the years, computer simulations have become indispensable in the study of enzyme mechanisms, with the integration of experimental and computational exploration now established as a holistic approach to gain deep insights into enzymatic catalysis. Numerous studies have demonstrated the power of computer simulations in characterizing reaction pathways, transition states, substrate selectivity, product distribution, and dynamic conformational changes for various enzymes. Nevertheless, significant challenges remain in investigating the mechanisms of complex multistep reactions, large-scale conformational changes, and allosteric regulation. Beyond mechanistic studies, computational enzyme modeling has emerged as an essential tool for computer-aided enzyme design and the rational discovery of covalent drugs for targeted therapies. Overall, enzyme design/engineering and covalent drug development can greatly benefit from our understanding of the detailed mechanisms of enzymes, such as protein dynamics, entropy contributions, and allostery, as revealed by computational studies. Such a convergence of different research approaches is expected to continue, creating synergies in enzyme research. This review, by outlining the ever-expanding field of enzyme research, aims to provide guidance for future research directions and facilitate new developments in this important and evolving field.
Biological life depends on motion, and this manifests itself in proteins that display motion over a formidable range of time scales spanning from femtoseconds vibrations of atoms at enzymatic transition states, all the way to slow domain motions occurring on micro to milliseconds. An outstanding challenge in contemporary biophysics and structural biology is a quantitative understanding of the linkages among protein structure, dynamics, and function. These linkages are becoming increasingly explorable due to conceptual and methodological advances. In this Perspective article, we will point toward future directions of the field of protein dynamics with an emphasis on enzymes. Research questions in the field are becoming increasingly complex such as the mechanistic understanding of high-order interaction networks in allosteric signal propagation through a protein matrix, or the connection between local and collective motions. In analogy to the solution to the "protein folding problem,"we argue that the way forward to understanding these and other important questions lies in the successful integration of experiment and computation, while utilizing the present rapid expansion of sequence and structure space. Looking forward, the future is bright, and we are in a period where we are on the doorstep to, at least in part, comprehend the importance of dynamics for biological function.
Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.
Enzyme function is often dependent on fluctuations between inactive and active structural ensembles. Adenylate kinase isolated from Escherichia coli (AK(e)) is a small phosphotransfer enzyme in which interconversion between inactive (open) and active (closed) conformations is rate limiting for catalysis. AK(e) has a modular three-dimensional architecture with two flexible substrate-binding domains that interact with the substrates AMP, ADP and ATP. Here, we show by using a combination of biophysical and mutagenic approaches that the interconversion between open and closed states of the ATP-binding subdomain involves partial subdomain unfolding/refolding in an otherwise folded enzyme. These results provide a novel and, possibly general, molecular mechanism for the switch between open and closed conformations in AK(e).
The biological function of proteins is critically dependent on dynamics inherent to the native structure. Such structural dynamics obey a predefined order and temporal timing to execute the specific reaction. Determination of the cooperativity of key structural rearrangements requires monitoring protein reactions in real time. In this work, we used time-resolved x-ray solution scattering (TR-XSS) to visualize structural changes in the Escherichia coli adenylate kinase (AdK) enzyme upon laser-induced activation of a protected ATP substrate. A 4.3-ms transient intermediate showed partial closing of both the ATP- and AMP-binding domains, which indicates a cooperative closing mechanism. The ATP-binding domain also showed local unfolding and breaking of an Arg131-Asp146 salt bridge. Nuclear magnetic resonance spectroscopy data identified similar unfolding in an Arg131Ala AdK mutant, which refolded in a closed, substrate-binding conformation. The observed structural dynamics agree with a “cracking mechanism” proposed to underlie global structural transformation, such as allostery, in proteins.
Cisplatin (cisPt), Pt(NH(3))(2)Cl(2), is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.
Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075: protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.
Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E. coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.
As a key molecule in biology, adenosine triphosphate (ATP) has numerous crucial functions in, for instance, energetics, post-translational modifications, nucleotide biosynthesis, and cofactor metabolism. Here, we have discovered an intricate interplay between the enzyme adenylate kinase and its substrate ATP. The side chain of an arginine residue was found to be an efficient sensor of the aromatic moiety of ATP through the formation of a strong cation−π interaction. In addition to recognition, the interaction was found to have dual functionality. First, it nucleates the activating conformational transition of the ATP binding domain and also affects the specificity in the distant AMP binding domain. In light of the functional consequences resulting from the cation−π interaction, it is possible that the mode of ATP recognition may be a useful tool in enzyme design.
ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3, which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle, where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and nonproductively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and nonproductive binding by the incorrect substrate bear a strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together, we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from nonlinearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These nonspecific interactions likely act to lower the available intracellular GTP and ATP concentrations and may have driven evolution of the Michaelis constants of NMP kinases accordingly.
Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.
The integration of highly efficient enzymatic catalysis with the solvation properties of ionic liquids for an environmentally friendly and efficient use of raw materials such as wood requires fundamental knowledge about the influence of relevant ionic liquids on enzymes. Switchable ionic liquids (SIL) are promising candidates for implementation of enzymatic treatments of raw materials. One industrially interesting SIL is constituted by monoethanol amine (MEA) and 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) formed with sulfur dioxide (SO2) as the coupling media (DBU-SO2-MEASIL). It has the ability to solubilize the matrix of lignocellulosic biomass while leaving the cellulose backbone intact. Using a novel 31P NMR-based real-time assay we show that this SIL is compatible with enzymatic catalysis because a model enzyme, adenylate kinase, retains its activity in up to at least 25 wt % of DBU-SO2-MEASIL. Thus this SIL appears suitable for, for example, enzymatic degradation of hemicellulose.
Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a 31P NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states.
Conformational change is regulating the biological activity of a large number of proteins and enzymes. Efforts in structural biology have provided molecular descriptions of the interactions that stabilize the stable ground states on the reaction trajectories during conformational change. Less is known about equilibrium thermodynamic stabilities of the polypeptide segments that participate in structural changes and whether the stabilities are relevant for the reaction pathway. Adenylate kinase (Adk) is composed of three subdomains: CORE, ATPlid, and AMPbd. ATPlid and AMPbd are flexible nucleotide binding subdomains where large-scale conformational changes are directly coupled to catalytic activity. In this report, the equilibrium thermodynamic stabilities of Adk from both mesophilic and hyperthermophilic bacteria were investigated using solution state NMR spectroscopy together with protein engineering experiments. Equilibrium hydrogen to deuterium exchange experiments indicate that the flexible subdomains are of significantly lower thermodynamic stability compared to the CORE subdomain. Using site-directed mutagenesis, parts of ATPlid and AMPbd could be selectively unfolded as a result of perturbation of hydrophobic clusters located in these respective subdomains. Analysis of the perturbed Adk variants using NMR spin relaxation and Cα chemical shifts shows that the CORE subdomain can fold independently of ATPlid and AMPbd; consequently, folding of the two flexible subdomains occurs independently of each other. Based on the experimental results it is apparent that the flexible subdomains fold into their native structure in a noncooperative manner with respect to the CORE subdomain. These results are discussed in light of the catalytically relevant conformational change of ATPlid and AMPbd.
Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.
Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies.
Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while 19F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be "atypical." On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.
Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16meso) and hyperthermophilic (S16thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔCp0) of 6.4 and 3.3 kJ mol− 1 K− 1, respectively. A strong linear correlation between ΔCp0 and melting temperature (Tm) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16meso folds through an apparent two-state model, whereas S16thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16thermo is more compact relative to S16meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16thermo that appears to account for the large difference in quantified ΔCp0 values and, in turn, parts of the observed extreme thermal stability of S16thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.
The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.
It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.
Allosteric regulation of protein activity is a fundamental principle for the temporal and spatial control of cellular function. Transfer of such principles to rational design of proteins will pave the way for biotechnological applications were control of a given process with external cues is desirable. Through a semi-rational and directed evolution approach we have been able to design a calcium dependent molecular switch with distinct “on” and “off” states decided by the presence and absence of calcium, respectively. The design was established in the context of protein-protein interactions with antibodies which is a massive biotechnological application linked to purification of therapeutic antibodies. Our approach was to introduce a randomized calcium binding loop into the C2 domain of Streptococcal Protein G. The large ensemble of different sequences was displayed on the surface of E. coli and subjected to selective pressures for binding to a human FAB in the presence but not in the absence of calcium. From this directed evolution experiment we discovered evolved variants that contained calcium switches with distinct “on” and “off” behavior towards FAB binding. The molecular mechanism underlying the calcium switch was discovered from quantification of both structure and dynamics with NMR spectroscopy. We found the designed protein was partially disordered in the absence of calcium, and that the disordered segment corresponded to the calcium loop and part of the FAB interaction surface of the parental C2 domain. In presence of calcium both the calcium binding loop and the FAB surface became fully structured and as a consequence the FAB binding activity was restored. Therefore, the calcium-switch in our designed protein is dictated by a “coupled folding and binding” mechanism, a principle that has evolved over and over again under natural selection in for instance intrinsically disordered proteins.
A fundamental question is how enzymes can accelerate chemical reactions. Catalysis is not only defined by actual chemical steps, but also by enzyme structure and dynamics. To investigate the role of protein dynamics in enzymatic turnover, we measured residue-specific protein dynamics in hyperthermophilic and mesophilic homologs of adenylate kinase during catalysis. A dynamic process, the opening of the nucleotide-binding lids, was found to be rate-limiting for both enzymes as measured by NMR relaxation. Moreover, we found that the reduced catalytic activity of the hyperthermophilic enzyme at ambient temperatures is caused solely by a slower lid-opening rate. This comparative and quantitative study of activity, structure and dynamics revealed a close link between protein dynamics and catalytic turnover.
Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AKeco) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turn-over rate is inversely proportional to the life-time of the active conformation. Therefore, structural plasticity is intimately cou-pled to enzymatic turn-over in AKeco. Here we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate, and in turn, the turn-over number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding sub-domains results in a population shift towards the open conformation and a resulting increase of kcat. Addition of the osmolyte TMAO to AKeco results in population shift towards the closed conformation and a significant reduction of kcat. The Michaelis constants (KM) scale with the change in kcat, which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, kcat and KM are mutually dependent and, in the case of AKeco, any perturbation that modulates kcat is mirrored with a proportional response in KM. Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affect enzymatic catalysis. From an evolutionary perspective our findings suggests that, for AKeco, there exists ample flexibility to obtain a specificity constant (kcat/KM) that commensurate with the exerted cellular selective pressure.