Free Energy Coupling Research Articles

Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs.

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNAleu for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, Δ(ΔG‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with 32Pα-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

Unconditionally stable, efficient and robust numerical simulation of isothermal compositional grading by gravity

The gravitational force has been considered as one of the most important factors leading to composition variation of multicomponent chemical species mixture in many industrial processes and natural phenomena. This has been largely studied through experimental and numerical modeling, especially in chemical processes and petroleum reservoir engineering. The modeling and simulation of dynamical process of composition variation under gravity is fundamentally important to understand the evolutionary process of petroleum reservoir formation and initial state. This work presents the dynamical modeling of composition variation in the framework of the modified Helmholtz free energy coupling with the realistic equations of state. An efficient, easy-to-implement, thermodyanmically consistent, and robust numerical scheme is proposed for the dynamical model. This scheme is rigorously proved to be unconditionally stable. The implementation is straightforward based on the single-component system and it is not required to choose a reference species for multicomponent fluids. For the multicomponent system of huge number of species, the proposed scheme allows to numerically compute the system of partial differential equations in a random order, which is called an “unbiased scheme” in this work. The current scheme is computationally efficient and saves computer memory. Several numerical examples are designed to verify the properties of the scheme.

Bridge-Mediated Charge Separation in Isomeric N-Annulated Perylene Diimide Dimers.

The possibility and rate of charge separation (CS) in donor-bridge-acceptor molecules mainly depend on two factors: electronic coupling and solvent effects. The question of how CS occurred in two identical chromophores is fundamental, as it is particularly interesting for potential molecular electronics applications and the photosynthetic reaction centers (RCs). Conjugated bridge definitely plays a crucial role in electronic coupling. To determine the bridge-mediated charge separation dynamics between the two identical chromophores, the isomeric N-annulated perylene diimide dimers (para-BDNP and meta-BDNP) with different conjugated bridge structures have been comparatively investigated in different solvents using femtosecond transient absorption spectra (fs-TA). It is found that the charge separation is disfavored in weak polar solvent, whereas direct spectroscopic signatures of radicals are observed in polar solvents, and the rate of charge separation increases as the solvent polarity increasing. To our surprise, the rate of charge separation in m-BDNP is more than an order of magnitude slower than that in p-BDNP, although there is a larger negative ΔGCS in m-BDNP. The slow CS rate that occurred in m-BDNP mainly results from the intrinsic destructive interference of the wave function through the meta-substituted bridge. The roles of solvent effects in free energy and electronic coupling for charge separation are further identified with quantum calculations.

Ligand Binding Free Energies with Adaptive Water Networks: Two-Dimensional Grand Canonical Alchemical Perturbations.

Computational methods to calculate ligand binding affinities are increasing in popularity, due to improvements in simulation algorithms, computational resources, and easy-to-use software. However, issues can arise in relative ligand binding free energy simulations if the ligands considered have different active site water networks, as simulations are typically performed with a predetermined number of water molecules (fixed N ensembles) in preassigned locations. If an alchemical perturbation is attempted where the change should result in a different active site water network, the water molecules may not be able to adapt appropriately within the time scales of the simulations—particularly if the active site is occluded. By combining the grand canonical ensemble (μVT) to sample active site water molecules, with conventional alchemical free energy methods, the water network is able to dynamically adapt to the changing ligand. We refer to this approach as grand canonical alchemical perturbation (GCAP). In this work we demonstrate GCAP for two systems; Scytalone Dehydratase (SD) and Adenosine A2A receptor. For both systems, GCAP is shown to perform well at reproducing experimental binding affinities. Calculating the relative binding affinities with a naïve, conventional attempt to solvate the active site illustrates how poor results can be if proper consideration of water molecules in occluded pockets is neglected. GCAP results are shown to be consistent with time-consuming double decoupling simulations. In addition, by obtaining the free energy surface for ligand perturbations, as a function of both the free energy coupling parameter and water chemical potential, it is possible to directly deconvolute the binding energetics in terms of protein–ligand direct interactions and protein binding site hydration.

Directing Charge Transfer in Quantum Dot Assemblies.

The optical and electronic properties of semiconductor quantum dots (QDs) make them attractive candidates for applications in photovoltaics, spintronics, photocatalysis, and optoelectronics. Understanding how to control the flow of charge in QD assemblies is essential for realizing novel applications. This Account explores some unique characteristics of charge transport in QD dyads, triads, and their assemblies. The emerging features of these assemblies that provide new opportunities to manipulate charge flow at the nanoscale are (1) cascading energy landscapes and band offsets to inhibit charge recombination, (2) electrostatic fields that direct charge flow through QD-QD and QD-conjugated polymer junctions, and (3) QD chirality and chiral imprinting that promotes vectorial electron and spin selective transport. Charge flow kinetics is determined by a combination of familiar electron transfer parameters (reaction free energy, reorganization energy, and electronic coupling), donor and acceptor electronic densities of states, and internal electric fields. Electron transfer and electronic structure theory, combined with kinetic modeling, place the measured kinetics of QD electron transfer donor-acceptor assemblies into a unified conceptual context. The experimental transfer rates measured in these systems depend upon structure and the internal electric fields that are present in the assemblies. A negatively charged donor and positively charged acceptor, for example, facilitates (inhibits) electron (hole) transfer, while an electric field of opposite orientation (reversal of charges) inhibits (promotes) electron (hole) transfer. These and other emerging rules that govern charge flow in NP assemblies provide a strategy to design the directionality and yield of interfacial charge transport. Chirality at the nanoscale can induce spin selective charge transport, providing new ways to direct charge (and spin) flow in QD assemblies. Magnetoresistance and magnetic conductive probe atomic force microscopy experiments show spin selective electron transport for chirally imprinted QD assemblies. Photoinduced electron transfer from achiral donor-QDs to chiral acceptor-QDs depends on the electron spin and chiroptical properties of the acceptor-QDs. These assemblies show transport characteristics that correlate with features of the QDs' circular dichroism spectra, presenting intriguing challenges to theory, and indicating that spectroscopic signatures may assist in the design and diagnosis of functional molecular assemblies. Theoretical and experimental studies of charge transport in well-defined QD assemblies are establishing design principles for vectorial charge transport and are also refining questions surrounding the mechanism and control of these processes. These intensified efforts are forging links between fundamental discoveries regarding mechanism and practical applications for these novel assembled nanostructures.

Nematic liquid crystals on curved surfaces: a thin film limit.

We consider a thin film limit of a Landau-de Gennes Q-tensor model. In the limiting process, we observe a continuous transition where the normal and tangential parts of the Q-tensor decouple and various intrinsic and extrinsic contributions emerge. The main properties of the thin film model, like uniaxiality and parameter phase space, are preserved in the limiting process. For the derived surface Landau-de Gennes model, we consider an L2-gradient flow. The resulting tensor-valued surface partial differential equation is numerically solved to demonstrate realizations of the tight coupling of elastic and bulk free energy with geometric properties.

Free Energy Coupling between DNA Bending and Base Flipping.

Free energy simulations are presented to probe the energetic coupling between DNA bending and the flipping of a central thymine in double stranded DNA 13mers. The energetics are shown to depend on the neighboring base pairs, and upstream C or T or downstream C tended to make flipping more costly. Flipping to the major groove side was generally preferred. Bending aids flipping, by pushing the system up in free energy, but for small and intermediate bending angles the two were uncorrelated. At higher bending angles, bending and flipping became correlated, and bending primed the system for base flipping toward the major groove. Flipping of the 6-4 pyrimidine-pyrimidone and pyrimidine dimer photoproducts is shown to be more facile than for undamaged DNA. For the damages, major groove flipping was preferred, and DNA bending was much facilitated in the 6-4 pyrimidine-pyrimidone damaged system. Aspects of the calculations were verified by structural analyses of protein-DNA complexes with flipped bases.

Between the Sheets: Inter-Subunit Backbone Interactions at AChR Neurotransmitter Binding Sites

The two neuromuscular AChR agonist-binding pockets are sandwiched between β-structures of α and e/δ/γ subunits. Within the global, channel-opening gating isomerization, a low-to-high affinity change for the agonist at each site serves to increase PO. There are two conserved, vicinal prolines at the end strand β5’ in each non-α subunit. We used single-channel electrophysiology to estimate energy changes caused by mutations here (γP120 and γP121), with regard to unliganded gating and the affinity change. For both prolines, all mutations had little or no effect on constitutive activity in all subunits. AFLR mutations of the first proline had no effect on the affinity change for ACh in e and δ subunits, but a modest effect in γ. At γP121, R and L substitutions had a large effect on the affinity change (>+3kcal/mol) but AGY were off less consequence (for all mutations the effect was smaller for choline than for ACh). Mutant cycle analyses were used to measure free energy coupling regarding the affinity change for ACh between γP121 and other residues near the binding pocket. γP121A interacts strongly with αG147A (strand β7) and αY198A (strand β10), modestly with αW149A (loop B) and αY190A (loop C) and not at all with αY93A (loop A) and αG153A (loop B). There was no coupling between γP121A and its neighbor γW55A (strand β1). The results suggest that strand β5’ in the γ subunit interacts with strands β7 and β10 in the α subunit, but less-so with residues in flexible loops that nonetheless influence affinity. Our working hypothesis is that β strands of the α and non-α subunits exchange energy during the affinity change for the agonist.

Effect of Crystal Meth and Ecstasy Enantiomers on Function of Dopamine Transporters

The complexities of the brain are hidden in the always active neuron communication. The propagation of the neuron signals is carried out by neurotransmitters. It is obvious why the signals activation is important but the signal quenching is just at important in proper brain function. The length of the stimuli and in turn the intensity are controlled by the neurotransmitter transporters.The clearing of the neurotransmitters from the synapse is the responsibility of transporters. Each neurotransmitter has its specific transporter. The main ones being Serotonin, Dopamine, and Norepinephrine Transporters (SERT, DAT, and NET). As a class of secondary transporters the sodium:neurotransmitter symporters utilize a sodium ion gradient to co-transport a neurotransmitter molecule against its gradient. The coupling of the ions favourable free energy to the unfavourable recycling of the neurotransmitters is the crucial step in deciphering the mechanism of transport.The interaction between the substrate and protein are key to proper transport. However these transporters are very common targets not only for the neurotransmitters but many medicinal and psychedelic drugs. Our focus is on exploring similarities between the substrates and what properties make them likely to target transporters. Also we wish to explore the binding differences experimentally observed in different enantiomers of methamphetamines (crystal meth) and 3,4-methylenedioxymethamphetamine (ecstasy). We have used homology modelling to model hDAT and hSERT in three different conformations (open-to-out, open-to-in, and occluded). These six structures will be used to explore the differences between the S and R enantiomers. Like many biological systems preference is given to one conformation over the other, with S enantiomer being the highly preferred one.

Serotonin and Dopamine Transporter Homology Models Provide a Look Inside the Active Site during Three Stages of Transport

The central nervous system is controlled by electrochemical impulses propagated through neurotransmitters, whose concentration and length of presence in the synapse determines the duration and the strength of the stimuli. Clearing of the neurotransmitters is the responsibility of specific transporters. The main ones being Serotonin, Dopamine, and Norepinephrine Transporters (SERT, DAT, and NET). As a class of secondary transporters the sodium: neurotransmitter symporters utilize a sodium ion gradient to co-transport a neurotransmitter molecule against its gradient. The coupling of the ions favourable free energy to the unfavourable recycling of the neurotransmitters is the crucial step in deciphering the mechanism of action. Our interests also expand into the substrate interaction with the transporter at the active site. Studying not just the interaction of the protein with the native substrate, but some known inhibitors will provide even more insight into the interactions at the active site. The inhibitors we are interested in are both of pharmaceutical and psychedelic origins. The interesting idea behind looking at both of these is the difference in addictiveness of one over the other. Different inhibitors will also have different modes of action and by exploiting these we can come up with more effective and less addictive drugs for treating many mood and neurodegenerative disorders. The first step in our approach will be to create hSERT and hDAT homology models using LeuT as a template. Each homology models will be modelled in three different states. The open-to-out, open-to-in, and fully occluded states will provide us with the “snap shots” during the transport. By analyzing the differences and similarities in ion coupling and substrate/inhibitor interactions clues to the overall mechanism will be obtained.

Cooperative DNA-Binding Effect through DNA Allostery

Allostery is well-documented for proteins but less recognized for DNA-protein interactions, in which DNA has been often considered as a mere template providing recognition sequences. Here we report that for two proteins bound on DNA at a separation of tens of base pairs, their DNA binding affinities can be significantly altered. This coupling effect oscillates between positive and negative cooperativity, depending on the separation distance between the two proteins on the DNA. With a DNA hairpin experiment, we provide definitive evidence for the structural basis of DNA allostery. We prove this effect is not due to protein-protein interactions but originates from the distortion of the inter-helical distance along the linker DNA. The oscillation has a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA, and a characteristic decay length of ∼15 base pairs. In the theoretical analysis, we elucidate the relation between the mechanical structural distortion of DNA induced by protein-binding and the free energy coupling measured thermodynamically, providing a complete picture for the origin of DNA allostery. The allosteric coupling between two DNA-bound proteins is found to be ubiquitous, regardless of proteins’ properties, implying its general roles in gene regulation. We demonstrate such DNA allostery affects gene expression levels in live E.coli cells. Pertinent to eukaryotic gene expression, we show that the binding affinity of a transcription factor depends on its separation from nearby nucleosomes. This work provides the first comprehensive study of allostery through DNA, with the understanding of its physical underpinning and ubiquity and biological relevance.

Character of the structural and magnetic phase transitions in the parent and electron-doped BaFe2As2compounds

We present a combined high-resolution x-ray diffraction and x-ray resonant magnetic scattering (XRMS) study of as-grown BaFe2As2. The structural/magnetic transitions must be described as a two-step process. At T_S = 134.5 K we observe the onset of a second-order structural transition from the high-temperature paramagnetic tetragonal structure to a paramagnetic orthorhombic phase, followed by a discontinuous step in the structural order parameter that is coincident with a first-order antiferromagnetic (AFM) transition at T_N = 133.75 K. These data, together with detailed high-resolution x-ray studies of the structural transition in lightly doped Ba(Fe{1-x}Co{x})2As2 and Ba(Fe{1-x}Rh{x})2As2 compounds, show that the structural and AFM transitions do, in fact, occur at slightly different temperatures in the parent BaFe2As2 compound, and evolve towards split secondorder transitions as the doping concentration is increased. We estimate the composition for the tricritical point for Co-doping and employ a mean-field approach to show that our measurements can be explained by the inclusion of an anharmonic term in the elastic free energy and magneto-elastic coupling in the form of an emergent Ising-nematic degree of freedom.

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. One structural model predicts sequential formation of ion pairs between the S4 gating charges and negative charges in neighboring S2 and S3 transmembrane segments during gating. Here, we show that paired cysteine substitutions for the third gating charge (R3) in S4 and D60 in S2 of the bacterial sodium channel NaChBac form a disulfide bond during activation, thus "locking" the S4 segment and inducing slow inactivation of the channel. Disulfide locking closely followed the kinetics and voltage dependence of activation and was reversed by hyperpolarization. Activation of D60C:R3C channels is favored compared with single cysteine mutants, and mutant cycle analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation.

Molecular dynamics simulation of ultrafast laser ablation of fused silica film

Ultrafast laser ablation of fused silica is studied using molecular dynamics simulations. Ionization and generation of free electrons, absorption of the laser energy by free electrons and energy coupling between free electrons and ions are considered. The BKS potential is applied and modified to describe molecular interactions and the effect of free electrons. Smooth particle mesh of the Ewald method (SPME) is adopted to calculate the Coulomb force. It is found that the electrostatic Coulomb force, which is caused by the ionization, plays an important role in the laser ablation process.

Structure of the Ebola Fusion Peptide in a Membrane-mimetic Environment and the Interaction with Lipid Rafts

The fusion peptide EBO(16) (GAAIGLAWIPYFGPAA) comprises the fusion domain of an internal sequence located in the envelope fusion glycoprotein (GP2) of the Ebola virus. This region interacts with the cellular membrane of the host and leads to membrane fusion. To gain insight into the mechanism of the peptide-membrane interaction and fusion, insertion of the peptide was modeled by experiments in which the tryptophan fluorescence and (1)H NMR were monitored in the presence of sodium dodecyl sulfate micelles or in the presence of detergent-resistant membrane fractions. In the presence of SDS micelles, EBO(16) undergoes a random coil-helix transition, showing a tendency to self-associate. The three-dimensional structure displays a 3(10)-helix in the central part of molecule, similar to the fusion peptides of many known membrane fusion proteins. Our results also reveal that EBO(16) can interact with detergent-resistant membrane fractions and strongly suggest that Trp-8 and Phe-12 are important for structure maintenance within the membrane bilayer. Replacement of tryptophan 8 with alanine (W8A) resulted in dramatic loss of helical structure, proving the importance of the aromatic ring in stabilizing the helix. Molecular dynamics studies of the interaction between the peptide and the target membrane also corroborated the crucial participation of these aromatic residues. The aromatic-aromatic interaction may provide a mechanism for the free energy coupling between random coil-helical transition and membrane anchoring. Our data shed light on the structural "domains" of fusion peptides and provide a clue for the development of a drug that might block the early steps of viral infection.

Conformational Analysis of the Electron-Transfer Kinetics across Oligoproline Peptides Using N,N-Dimethyl-1,4-benzenediamine Donors and Pyrene-1-sulfonyl Acceptors

Photoinduced intramolecular charge separation across proline-bridged donor-acceptor complexes of the type Pyr-(Pro)n-DMPD (where Pyr=pyrene-1-sulfonyl and DMPD=N,N-dimethyl-1,4-phenylenediamine) was studied. The steady-state emission spectrum for n=0, 1, 2, 3 showed an increase in emission intensity with the number of proline residues. Time-dependent emission measured by streak camera showed increasing emission signal amplitude with increasing n, along with a decrease in decay rate. In all these studies, Pyr-Pro was used as a control complex for the decay of the excited pyrene acceptor moiety without the donor DMPD. Detailed photon counting experiments carried out in DMF/water, DMF, and toluene showed single-exponential kinetics for n=0, 1 and multiexponential kinetics for n=2, 3. Rate constants observed in DMF are for n=0, k=approximately 5x10(10) s(-1); n=1, k=9.70x10(8) s(-1); n=2, k=35.9x10(8) s(-1) (70%) and 5.58x10(8) s(-1) (30%); and n=3, k=16.6x10(8) s(-1) (55%) and 3.87x10(8) s(-1) (45%). These results show that a significant percentage of the n=2 and n=3 molecules undergo faster electron transfer than for the n=1 case. Conformational analysis for Pyr-(Pro)n-DMPD molecules in water showed that whereas only one conformation is possible for n=1, eight are possible for n=2, and 32 are possible for n=3. Calculation of the free energy and electronic coupling for these conformers in water showed that only a few of these conformations have the appropriate energy and electronic coupling to be observed in the experimental time window from 20 ps to 20 ns. Assignment of the conformers undergoing electron transfer in Pyr-(Pro)n-DMPD for n=2 and 3 was based on the values for the n=1 case, for which the measured rate constant is approximately 10(9) s(-1) and the calculated electronic coupling matrix element Hda is 297 cm(-1). The similarity in ground state energy between the cis and trans conformers for n=2 and 3, their use in aqueous-organic and organic solvents, and the nature of the Pyr and DMPD acceptor and donor groups could be contributing causes for the multiexponential kinetics, which was not observed for the metal ion derivatives of proline peptides studied earlier in aqueous solution.

Molecular dynamics simulation of ultrafast laser ablation of fused silica

Ultrafast laser ablation of fused silica is studied using molecular dynamics simulations. Ionization and generation of free electrons, absorption of the laser energy by free electrons and energy coupling between free electrons and ions are considered. The BKS potential is used to describe molecular interactions and is modified to include the effect of free electrons. Temperature, density, and material removal are computed, and the thermal and non-thermal mechanisms of ultrafast laser ablation of fused silica are discussed.

Predicting coupling limits from an experimentally determined energy landscape

Repeat proteins are composed of tandem structural modules in which close contacts do not extend beyond adjacent repeats. Despite the local nature of these close contacts, repeat proteins often unfold as a single, highly coupled unit. Previous studies on the Notch ankyrin domain suggest that this lack of equilibrium unfolding intermediates results both from stabilizing interfaces between each repeat and from a roughly uniform distribution of stability across the folding energy landscape. To investigate this idea, we have generated 15 variants of the Notch ankyrin domain with single and multiple destabilizing substitutions that make the energy landscape uneven. By applying a free energy additivity analysis to these variants, we quantified the destabilization threshold over which repeats 6 and 7 decouple from repeats 1-5. The free energy coupling limit suggested by this additivity analysis ( approximately 4 kcal/mol) is also reflected in m-value analysis and in differences among equilibrium unfolding transitions as monitored by CD versus fluorescence for all 15 variants. All of these observations are quantitatively predicted by analyzing the response of the experimentally determined energy landscape to increasing unevenness. These results highlight the importance of a uniform distribution of local stability in achieving cooperative unfolding.

Role of the Streptokinase α-Domain in the Interactions of Streptokinase with Plasminogen and Plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg </=2-fold, but surprisingly, rendered both interactions near totally LBS-dependent. The LBS-independent affinity of SK for [Lys]Pm, 3000-fold higher compared with [Lys]Pg, was reduced dramatically by alpha-domain truncation. Thermodynamic analysis demonstrates that the SK alpha-domain contributes substantially to affinity for all Pg/Pm species solely through LBS-independent interactions, and that the higher affinity of SK for [Lys]Pm compared with [Lys]Pg involves all three SK domains. The residual affinity of the SK betagamma-fragment for all Pg/Pm species was increased by an enhanced contribution to complex stability from LBS-dependent interactions or free energy coupling between LBS-dependent and -independent interactions. Redistribution of the free energy contributions accompanying alpha-domain truncation demonstrates the interdependence of SK domains in stabilizing the SK-Pg/Pm complexes. The flexible segments connecting the SK alpha, beta, and gamma domains allow their rearrangement into a distinctly different bound conformation accompanying loss of the constraint imposed by interactions of the alpha-domain.

Magnesium-dependent ATPase Activity and Cooperativity of Magnesium Chelatase from Synechocystis sp. PCC6803

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg(2+) activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of approximately 15 MgATP(2-) and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a DeltaG degrees ' of 25-33 kJ mol(-1). Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steady-state kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K(DIX) (where D(IX) represents deuteroporphyrin IX) is estimated to be 3.20 microm, and K(MgATP)(2-) is 0.45 mm. k(cat) for chelation is estimated to be 0.8 min(-1), suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP(2-) and D(IX), possibly as a result of a magnesium-triggered conformational change.