84 research outputs found

    On the relationship between thermal stability and catalytic power of enzymes

    Get PDF
    The  possible  relationship  between  the  thermal  stability  and  the  catalytic  power  of  enzymes  is  of   great  current  interest.  In  particular,  it  has  been  suggested  that  thermophilic  or  hyperthermophilic   (Tm)   enzymes   have   lower   catalytic   power   at   a   given   temperature   than   the   corresponding   mesophilic   (Ms)   enzymes,   because   the   thermophilic   enzymes   are   less   flexible   (assuming   that   flexibility   and   catalysis   are   directly   correlated).   These   suggestions   presume   that   the   reduced   dynamics   of   the   thermophilic   enzymes   is   the   reason   for   their   reduced   catalytic   power.   The   present  paper  takes  the  specific  case  of  dihydrofolate  reductase  (DHFR) and explores the validity of the above argument by simulation approaches. It is found that the Tm enzymes have restricted motions in the direction of the folding coordinate, but this is not relevant to the chemical process, since the motions along the reaction coordinate are perpendicular to the folding motions. Moreover, it is shown that the rate of the chemical reaction is determined by the activation barrier and the corresponding reorganization energy, rather than by dynamics or flexibility in the ground state. In fact, as far as flexibility is concerned, we conclude that the displacement along the reaction coordinate is larger in the Tm enzyme than in the Ms enzyme and that the general trend in enzyme catalysis is that the best catalyst involves less motion during the reaction than the less optimal catalyst. The relationship between thermal stability and catalysis appears to reflect the fact that in order to obtain small electrostatic reorganization energy it is necessary to invest some folding energy in the overall preorganization process. Thus, the optimized catalysts are less stable. This trend is clearly observed in the DHFR case

    Role of Microsolvation and Quantum Effects in the Accurate Prediction of Kinetic Isotope Effects: The Case of Hydrogen Atom Abstraction in Ethanol by Atomic Hydrogen in Aqueous Solution

    Get PDF
    Hydrogen abstraction from ethanol by atomic hydrogen in aqueous solution is studied using two theoretical approaches: the multipath variational transition state theory (MP-VTST) and a path-integral formalism in combination with free-energy perturbation and umbrella sampling (PI-FEP/UM). The performance of the models is compared to experimental values of H kinetic isotope effects (KIE). Solvation models used in this study ranged from purely implicit, via mixed–microsolvation treated quantum mechanically via the density functional theory (DFT) to fully explicit representation of the solvent, which was incorporated using a combined quantum mechanical-molecular mechanical (QM/MM) potential. The effects of the transition state conformation and the position of microsolvating water molecules interacting with the solute on the KIE are discussed. The KIEs are in good agreement with experiment when MP-VTST is used together with a model that includes microsolvation of the polar part of ethanol by five or six water molecules, emphasizing the importance of explicit solvation in KIE calculations. Both, MP-VTST and PI-FEP/UM enable detailed characterization of nuclear quantum effects accompanying the hydrogen atom transfer reaction in aqueous solutionThis work was partially supported by the National Science Center in Poland (Sonata BIS grant UMO-2014/14/E/ST4/00041) and in part by PLGrid Infrastructure (Poland). S.K. acknowledges the Erasmus+ programme within which his 3-month project conducted at the University of Santiago de Compostela was possible. A.F-.R. thanks the Consellería de Cultura, Educación e Ordenación Universitaria (Axuda para Consolidación e Estructuración de unidades de investigación competitivas do Sistema Universitario de Galicia, Xunta de Galicia ED431C 2017/17 & Centro singular de investigación de Galicia acreditación 2016-2019, ED431G/09) and the European Regional Development Fund (ERDF). D.F-.C. also thanks Xunta de Galicia for financial support through a postdoctoral grantS

    On the Preorganization of the Active Site of Choline Oxidase for Hydride Transfer and Tunneling Mechanism

    Get PDF
    Choline oxidase catalyzes the two-step oxidation of choline to glycine betaine, one of limited osmoprotectants, with the formation of betaine aldehyde as an enzyme bound intermediate. Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism to withstand hyperosmolarity and elevated temperatures. This makes the genetic engineering of relevant plants which lack the property of salt accumulation of economic interest, and the biosynthetic pathway of the osmolyte a potential drug target in microbial infections. The reaction of alcohol oxidation occurs via a hydride ion tunneling transfer from the substrate donor to a flavin acceptor within a highly preorganized active site environment in which choline and FAD are in a rigidly close proximity. In this dissertation, factors contributing to the enzyme-substrate preorganization which is required for the hydride ion tunneling reaction mechanism in choline oxidase have been investigated. Crystallographic studies of wild-type choline oxidase revealed a covalent linkage between C8M atom of the FAD isoalloxazine ring and the N(3) atom of the side chain of a histidine at position 99, and a solvent excluded cavity in the substrate binding domain containing glutamic acid at position 312 as the only negatively charged amino acid residue in the active site of the enzyme. The role of the histidine residue and the contribution of the 8á-N(3)-histidyl covalent linkage of the flavin cofactor to the reaction of alcohol oxidation was investigated in a variant form of choline oxidase in which the histidine residue was replaced with an asparagine. The role of the glutamate residue and the importance of the spatial location of the negative charge at position 312 was investigated in variant forms of choline oxidase in which the negatively charged residue was replaced with glutamine and aspartate. Mechanistic data obtained for the variant enzymes and their comparison to previous data obtained for wild-type choline oxidase are consistent with the residues at positions 99 and 312 being important for relative positioning of the hydride ion donor and acceptor. The residues are important for the enzyme-substrate preorganization that is required for the hydride tunneling reaction in choline oxidase

    Effect of organic co-solvents on the NMR chemical shifts and dynamics of dihydrofolate reductase from E. coli

    Get PDF
    Enzymes are large biological molecules that can exist in many different states and each state may have multiple conformations. The probability of finding an enzyme in a particular state is governed by the free energy landscape and the energy barriers between the different states. The energy landscape of an enzyme can be perturbed through making changes to the external conditions such as the reaction medium, which can cause a shift in the delicate equilibrium between states. Experimental evidence suggests that the model for environmentally coupled tunnelling is inconsistent for catalysis by dihydrofolate reductase (DHFR) from E coli.. It is proposed that catalysis is governed by electrostatics and characterised by populations of sub-states with distinct conformations and kinetics. Here, using solution NMR spectroscopy, the enzyme is probed at the point of the chemical reaction step by subjecting a mimic of the Michaelis complex (EcDHFR: NADP+: folate) to organic co-solvents in order to perturb the energy landscape of the protein by altering the viscosity and dielectric constant of the enzymatic reaction medium. The chemical shift assignment of the Michaelis complex mimic under standard buffer conditions, in the presence of 17% methanol and 17 % glycerol co-solvents (to effect almost isodielectric mediums that differ in viscosity) is reported. The majority of atoms in the protein complex in the presence of 17% glycerol and 17% methanol show small chemical shift perturbations (Chapter 4.0). The ps-ns relaxation dynamics of the Michaelis complex mimic were measured via solution NMR at 600 MHz and 900 MHz in the presence of co-solvents to determine any alterations in the timescale of dynamics on this timescale. The results indicate slightly increased mobility of some residues in loop regions in the presence of methanol and glycerol co-solvents and slightly decreased mobility of some residues in areas of defined secondary structure (Chapter 5.0)

    Getting Heavy: An Exploration into the Effects of D2O and High Hydrostatic Pressure on R67 Dihydrofolate Reductase

    Get PDF
    Chromosomal dihydrofolate reductase (DHFR) enzymatically reduces dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. R67 DHFR is an R-plasmid encoded enzyme that confers resistance to trimethoprim (TMP), an antibacterial drug. It shares no structural homology with TMP targeted, chromosomal DHFRs. Previous osmolyte studies in our lab have indicated that DHF binding to R67 DHFR is accompanied by water uptake and NADPH binding is accompanied by water release. These data suggest that water plays a role in balancing the binding affinity. This may happen as R67 DHFR has a generalized binding surface and may need differential water effects to accommodate both ligands. To further examine this hypothesis, we collect binding and steady state kinetic data using hydrostatic pressure. Increasing hydrostatic pressure hydrates molecules and can essentially test the effect of increasing water concentration upon binding. Hydrostatic pressure can also affect the volume of the active site as well. An activation volume, defined as the change in molar volume associated with the ternary E-NADPH-DHF complex going to the transition state, can be determined from a plot of the natural log of kcat vs pressure. The slope of this line is equal to - Δ[delta]V/RpT. A small slope giving an activation volume of - 1.03 ± 0.9 cm3/mol is observed until 200 Mpa. A second slope describing the effect of pressure from 200 Mpa to 500 Mpa on the activation volume was equal to 8.06 ± 0.8 cm3/mol. Positive activation volumes indicate that the rate-limiting step described accompanies a protein volume increase. As water reorganization may be playing a role in binding of both substrate and cofactor, studies using isothermal titration calorimetry in both H2O and D2O were utilized to determine the enthalpy of solvent reorganization. The observed enthalpy of the interaction between protein and substrate can be broken up into Δ[delta]Hi (enthalpy of the interaction) and Δ[delta]Hs (enthalpy of solvent reorganization). Since the enthalpy of a hydrogen bond in D2O is approximately 10% greater than in H2O, the ∆[delta]Hs can be estimated

    Contribution of Water and Energetics of Ligand Binding in the Catalytic Mechanism of R67 Dihydrofolate Reductase

    Get PDF
    R67 dihydrofolate reductase (DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate (DHF) to produce tetrahydrofolate (THF). The enzyme is a homotetramer and its 222 symmetry allows for binding of both ligands to a single active site pore. A productive ternary complex is formed by the binding of one molecule of DHF and NADPH and inter-ligand cooperativity has been suggested to be essential for binding and catalysis. To gain further insight into the thermodynamics involved in the ground state and the transition state, temperature dependent studies on DHF binding and catalysis were performed. It was observed that binding of both NADPH and DHF is enthalpy driven. From van’t Hoff plots, the change in enthalpy, entropy and free energy for NADPH binding to R67 DHFR in the ground state were determined. Similarly, the thermodynamics of DHF binding to the R67 DHFR-NADPH complex in the ground state were determined. Arrhenius plots were also employed to study the energetics of the transition state. A comparison of TdeltaS values (for DHF binding to R67 DHFR-NADPH complex) in both ground state and transition state indicates that TdeltaS is more negative in the transition state (–11.3 kcal/mol) as compared to the ground state (–5.4 kcal/mol). This indicates a reorientation of the substrate in the transition state. The role of water in DHF and NADPH binding to R67 DHFR was also investigated. For this, the effect of osmotic pressure on the Ka /Km of ligand binding, as well as the kcat of the reaction was studied. It was observed that the kcat of the reaction was not significantly affected, while the binding of ligands was affected with increasing osmolality. Specifically, binding of NADPH tightened as osmolality increased, while binding of DHF weakened with increasing osmolality, suggesting release of water upon NADPH binding and an uptake of water on DHF binding. Results from in vivo experiments on E.coli cells containing wild type and mutant clones of R67 DHFR were also consistent with in vitro experiments, suggesting that water is involved in ligand binding to R67 DHFR
    corecore