An approach to rational ligand-design based on a thermodynamic analysis

Thermodynamic analysis is an effective tool in screening of lead-compounds for development of potential drug candidates. In most cases, a ligand achieve high affinity and specificity to a target protein by means of both favorable enthalpy and entropy terms, which can be reflected in binding profiles of Isothermal Titration Calorimetry (ITC). A favorable enthalpy change suggests the contribution of noncovalent contacts such as hydrogen bonding and van der Waals interaction between a ligand and its target protein. In general, optimization of binding enthalpy is more difficult than that of entropies in ligand-design; therefore, it is desirable to choose firstly a lead-compound based on its binding enthalpic gain. In this paper, we demonstrate the utility of thermodynamic approach to ligand screening using anti-ciguatoxin antibody 10C9 as a model of a target protein which possesses a large hydrophobic pocket. As a result of this screening, we have identified three compounds that could bind to the antigen-binding pocket of 10C9 with a few kcal/mol of favorable binding enthalpy. Comparison of their structure with the proper antigen ciguatoxin CTX3C revealed that 10C9 rigorously identifies their cyclic structure and a characteristic hydroxyl group. ITC measurement might be useful and powerful for a rational ligand screening and the optimization of the ligand; the enthalpic gain is an effective index for ligand-design studies.     

Energetics of ligand binding to human glutathione transferase A1-1: Tyr-9 associated localisation of the C-terminal helix is ligand-dependent

A C-terminal helix (α9) adjacent to the active site on each subunit is a structural feature unique to the alpha isoform of glutathione transferases which contributes to the catalytic and ligandin functions of the enzyme. The ionisation state of Tyr-9, a residue critical to catalysis, influences α9 dynamics, although the mechanism is poorly understood. In this study, isothermal titration calorimetry was used to probe the binding energetics of G-site (glutathione and glutathione sulfonate) and H-site (ethacrynic acid) ligands to wild-type and a Y9F mutant of human glutathione transferase A1-1. Although previous studies have reported a favourable entropic component to the binding of conjugates occupying both sites, our data reveal that ligand binding is enthalpically driven when either the G- or H-site is occupied independently. Also, heat capacity changes demonstrate that α9 is fully localised by H-site but not G-site occupation. The Tyr-9 hydroxyl group contributes significantly to ligand binding energetics, although the effect differs between the two binding sites. G-site binding is made slightly enthalpically more favourable and entropically less favourable by the Y9F mutation. Binding to the H-site is more dramatically affected, with the K(d) for ethacrynic acid increasing 5 fold despite a more favourable ΔS. The heat capacity change is more negative for G-site binding in the absence of the Tyr-9 hydroxyl (ΔΔC(p)=-0.73 kJ mol(-1) K(-1)), but less negative for H-site binding to the Y9F mutant (ΔΔC(p)=0.63 kJ mol(-1) K(-1)). This suggests that the relationship between Tyr-9 and α9 is not independent of the ligand. Rather, Tyr-9 appears to function in orienting the ligand optimally for α9 closure.     

Structural and thermodynamic study on aldose reductase: nitro-substituted inhibitors with strong enthalpic binding contribution

To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. In order to identify novel lead compounds, a virtual screening (VS) was performed successfully suggesting carboxylate-type inhibitors of sub-micromolar to micromolar affinity. Here, we combine a structural characterization of the binding modes observed by X-ray crystallography with isothermal titration calorimetry (ITC) measurements providing insights into the driving forces of inhibitor binding, particularly of the first leads from VS. Characteristic features of this novel inhibitor type include a carboxylate head group connected via an alkyl spacer to a heteroaromatic moiety, which is linked to a further nitro-substituted aromatic portion. The crystal structures of two enzyme-inhibitor complexes have been determined at resolutions of 1.43 A and 1.55 A. Surprisingly, the carboxylic group of the most potent VS lead occupies the catalytic pocket differently compared to the interaction geometry observed in almost all other crystal structures with structurally related ligands and obtained under similar conditions, as an interstitial water molecule is picked up upon ligand binding. The nitro-aromatic moiety of both leads occupies the specificity pocket of the enzyme, however, adopting a different geometry compared to the docking prediction: unexpectedly, the nitro group binds to the bottom of the specificity pocket and provokes remarkable induced-fit adaptations. A peptide group located at the active site orients in such a way that H-bond formation to one nitro group oxygen atom is enabled, whereas a neighbouring tyrosine side-chain performs a slight rotation off from the binding cavity to accommodate the nitro group. Identically constituted ligands, lacking this nitro group, exhibit an affinity drop of one order of magnitude. In addition, thermodynamic data suggest a strongly favourable contribution to binding enthalpy in case the inhibitor is equipped with a nitro group at the corresponding position. To further investigate this phenomenon, we determined crystal structures and thermodynamic data of two similarly constituted IDD-type inhibitors addressing the specificity pocket with either a nitro or halogen-substituted aromatic moiety. As these data suggest, the nitro group provokes the enthalpic contribution, in addition to the H-bond mentioned above, by accepting two "non-classical" H-bonds donated by the aromatic tyrosine side-chain. In summary, this study provides the platform for further structure-guided design hypotheses of novel drug candidates with higher affinity and selectivity.     

Conformational selection through electrostatics: Free energy simulations of GTP and GDP binding to archaeal initiation factor 2

Archaeal Initiation Factor 2 is a GTPase involved in protein biosynthesis. In its GTP-bound, "ON" conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and their dependence on the conformational state, molecular dynamics free energy simulations were performed. The ON state specificity was predicted to be weak, with a GTP/GDP binding free energy difference of -1 kcal/mol, favoring GTP. The OFF state specificity is larger, 4 kcal/mol, favoring GDP. The overall effects result from a competition among many interactions in several complexes. To interpret them, we use a simpler, dielectric continuum model. Several effects are robust with respect to the model details. Both nucleotides have a net negative charge, so that removing them from solvent into the binding pocket carries a desolvation penalty, which is large for the ON state, and strongly disfavors GTP binding compared to GDP. Short-range interactions between the additional GTP phosphate group and ionized sidechains in the binding pocket offset most, but not all of the desolvation penalty; more distant groups also contribute significantly, and the switch 1 loop only slightly. The desolvation penalty is lower for the more open, wetter OFF state, and the GTP/GDP difference much smaller. Short-range interactions in the binding pocket and with more distant groups again make a significant contribution. Overall, the simulations help explain how conformational selection is achieved with a single phosphate group.     

Energetic analysis of binding of progesterone and 5 beta-androstane-3,17-dione to anti-progesterone antibody DB3 using molecular dynamics and free energy calculations.

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the energetics of the binding of progesterone (PRG) and 5 beta-androstane-3,17-dione (5AD) to anti-PRG antibody DB3. Although the two steroids bind to DB3 in different orientations, their binding affinities are of the same magnitude, 1 nM for PRG and 8 nM for 5AD. The calculated relative binding free energy of the steroids, 8.8 kJ/mol, is in fair agreement with the experimental energy, 5.4 kJ/mol. In addition, computational alanine scanning was applied to study the role of selected amino acid residues of the ligand-binding site on the steroid cross-reactivity. The electrostatic and van der Waals components of the total binding free energies were found to favour more the binding of PRG, whereas solvation energies were more favourable for the binding of 5AD. The differences in the free energy components are due to the binding of the A rings of the steroids to different binding pockets: PRG is bound to a pocket in which electrostatic antibody-steroid interactions are dominating, whereas 5AD is bound to a pocket in which van der Waals and hydrophobic interactions dominate.     

Thermodynamic consequences of disrupting a water-mediated hydrogen bond network in a protein:pheromone complex

The mouse pheromones (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT) and 6-hydroxy-6-methyl-3-heptanone (HMH) bind into an occluded hydrophobic cavity in the mouse major urinary protein (MUP-1). Although the ligands are structurally unrelated, in both cases binding is accompanied by formation of a similar buried, water-mediated hydrogen bond network between the ligand and several backbone and side chain groups on the protein. To investigate the energetic contribution of this hydrogen bond network to ligand binding, we have applied isothermal titration calorimetry to measure the binding thermodynamics using several MUP mutants and ligand analogs. Mutation of Tyr-120 to Phe, which disrupts a hydrogen bond from the phenolic hydroxyl group of Tyr-120 to one of the bound water molecules, results in a substantial loss of favorable binding enthalpy, which is partially compensated by a favorable change in binding entropy. A similar thermodynamic effect was observed when the hydrogen bonded nitrogen atom of the heterocyclic ligand was replaced by a methyne group. Several other modifications of the protein or ligand had smaller effects on the binding thermodynamics. The data provide supporting evidence for the role of the hydrogen bond network in stabilizing the complex.     

Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: Binding of phosphonamidate to the S1'-pocket of thermolysin

Background

Prerequisite for the design of tight binding protein inhibitors and prediction of their properties is an in-depth understanding of the structural and thermodynamic details of the binding process. A series of closely related phosphonamidates was studied to elucidate the forces underlying their binding affinity to thermolysin. The investigated inhibitors are identical except for the parts penetrating into the hydrophobic S₁'-pocket.

Methods

A correlation of structural, kinetic and thermodynamic data was carried out by X-ray crystallography, kinetic inhibition assay and isothermal titration calorimetry.

Results and conclusions

Binding affinity increases with larger ligand hydrophobic P₁'-moieties accommodating the S₁'-pocket. Surprisingly, larger P₁'-side chain modifications are accompanied by an increase in the enthalpic contribution to binding. In agreement with other studies, it is suggested that the release of largely disordered waters from an imperfectly hydrated pocket results in an enthalpically favourable integration of these water molecules into bulk water upon inhibitor binding. This enthalpically favourable process contributes more strongly to the binding energetics than the entropy increase resulting from the release of water molecules from the S₁'-pocket or the formation of apolar interactions between protein and inhibitor.

General significance

Displacement of highly disordered water molecules from a rather imperfectly hydrated and hydrophobic specificity pocket can reveal an enthalpic signature of inhibitor binding.     

Implications of the ligandin binding site on the binding of non-substrate ligands to Schistosoma japonicum-glutathione transferase

The binding interactions between dimeric glutathione transferase from Schistosoma japonicum (Sj26GST) and bromosulfophthalein (BS) or 8-anilino-1-naphthalene sulfonate (ANS) were characterised by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Both ligands inhibit the enzymatic activity of Sj26GST in a non-competitive form. A stoichiometry of 1 molecule of ligand per mole of dimeric enzyme was obtained for the binding of these ligands. The affinity of BS is higher (K(d)=3.2 microM) than that for ANS (K(d)=195 microM). The thermodynamic parameters obtained by calorimetric titrations are pH-independent in the range of 5.5 to 7.5. The interaction process is enthalpically driven at all the studied temperatures. This enthalpic contribution is larger for the ANS anion than for BS. The strongly favourable enthalpic contribution for the binding of ANS to Sj26GST is compensated by a negative entropy change, due to enthalpy-entropy compensation. DeltaG degrees remains almost invariant over the temperature range studied. The free energy change for the binding of BS to Sj26GST is also favoured by entropic contributions at temperatures below 32 degrees C, thus indicating a strong hydrophobic interaction. Heat capacity change obtained for BS (DeltaC(p) degrees =(-580.3+/-54.2) cal x K(-1) mol(-1)) is twofold larger (in absolute value) than for ANS (DeltaC(p) degrees =(-294.8+/-15.8) cal x K(-1) mol(-1)). Taking together the thermodynamic parameters obtained for these inhibitors, it can be argued that the possible hydrophobic interactions in the binding of these inhibitors to L-site must be accompanied by other interactions whose contribution is enthalpic. Therefore, the non-substrate binding site (designed as ligandin) on Sj26GST may not be fully hydrophobic.     

Think Twice: Understanding the High Potency of Bis(phenyl)methane Inhibitors of Thrombin

Successful design of potent and selective protein inhibitors, in terms of structure-based drug design, strongly relies on the correct understanding of the molecular features determining the ligand binding to the target protein. We present a case study of serine protease inhibitors with a bis(phenyl)methane moiety binding into the S3 pocket. These inhibitors bind with remarkable potency to the active site of thrombin, the blood coagulation factor IIa. A combination of X-ray crystallography and isothermal titration calorimetry provides conclusive insights into the driving forces responsible for the surprisingly high potency of these inhibitors. Analysis of six well-resolved crystal structures (resolution 1.58-2.25 Å) along with the thermodynamic data allows an explanation of the tight binding of the bis(phenyl)methane inhibitors. Interestingly, the two phenyl rings contribute to binding affinity for very different reasons — a fact that can only be elucidated by a structure-based approach. The first phenyl moiety occupies the hydrophobic S3 pocket, resulting in a mainly entropic advantage of binding. This observation is based on the displacement of structural water molecules from the S3 pocket that are observed in complexes with inhibitors that do not bind in the S3 pocket. The same classic hydrophobic effect cannot explain the enhanced binding affinity resulting from the attachment of the second, more solvent-exposed phenyl ring. For the bis(phenyl)methane inhibitors, an observed adaptive rotation of a glutamate residue adjacent to the S3 binding pocket attracted our attention. The rotation of this glutamate into salt-bridging distance with a lysine moiety correlates with an enhanced enthalpic contribution to binding for these highly potent thrombin binders. This explanation for the magnitude of the attractive force is confirmed by data retrieved by a Relibase search of several thrombin-inhibitor complexes deposited in the Protein Data Bank exhibiting similar molecular features.Special attention was attributed to putative changes in the protonation states of the interaction partners. For this purpose, two analogous inhibitors differing mainly in their potential to change the protonation state of a hydrogen-bond donor functionality were compared. Buffer dependencies of the binding enthalpy associated with complex formation could be traced by isothermal titration calorimetry, which revealed, along with analysis of the crystal structures (resolution 1.60 and 1.75 Å), that a virtually compensating proton interchange between enzyme, inhibitor and buffer is responsible for the observed buffer-independent thermodynamic signatures.     

Stabilization of anionic and neutral forms of a fluorophoric ligand at the active site of human carbonic anhydrase I

We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15 Å deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme–ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn²⁺ cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2 kcal/mol (ΔΔG°) and 0.89 kcal/mol (ΔΔG^‡) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed.     

Crystal structure of a recombinant anti-estradiol Fab fragment in complex with 17beta -estradiol.

The crystal structure of a Fab fragment of an anti-17beta-estradiol antibody 57-2 was determined in the absence and presence of the steroid ligand, 17beta-estradiol (E2), at 2.5 and 2.15-A resolutions, respectively. The antibody binds the steroid in a deep hydrophobic pocket formed at the interface between the variable domains. No major structural rearrangements take place upon ligand binding; however, a large part of the heavy chain variable domain near the binding pocket is unusually flexible and is partly stabilized when the steroid is bound. The nonpolar steroid skeleton of E2 is recognized by a number of hydrophobic interactions, whereas the two hydroxyl groups of E2 are hydrogen-bonded to the protein. Especially, the 17-hydroxyl group of E2 is recognized by an intricate hydrogen bonding network in which the 17-hydroxyl itself forms a rare four-center hydrogen bond with three polar amino acids; this hydrogen bonding arrangement accounts for the low cross-reactivity of the antibody with other estrogens such as estrone. The CDRH3 loop plays a prominent role in ligand binding. All the complementarity-determining regions of the light chain make direct contacts with the steroid, even CDRL2, which is rarely directly involved in the binding of haptens.     

Heat capacity effects in protein folding and ligand binding: a re-evaluation of the role of water in biomolecular thermodynamics

Large "anomalous" heat capacity (DeltaC(p)) effects are a common feature of the thermodynamics of biomolecular interactions in aqueous solution and, as a result of the improved facility for direct calorimetric measurements, there is a growing body of experimental data for such effects in protein folding, protein-protein and protein-ligand interactions. Conventionally such heat capacity effects have been ascribed to hydrophobic interactions, and there are some remarkably convincing demonstrations of the usefulness of this concept. Nonetheless, there is also increasing evidence that hydrophobic interactions are not the only possible source of such effects. Here we re-evaluate the possible contributions of other interactions to the heat capacity changes to be expected for cooperative biomolecular folding and binding processes, with particular reference to the role of hydrogen bonding and solvent water interactions. Simple models based on the hydrogen-bonding propensity of water as a function of temperature give quantitative estimates of DeltaC(p) that compare well with experimental observations for both protein folding and ligand binding. The thermodynamic contribution of bound waters in protein complexes is also estimated. The prediction from simple lattice models is that trapping of water in a complex should give more exothermic binding (DeltaDeltaH-6 to -12 kJ mol(-1)) with lower entropy (DeltaDeltaS(0) approximately -11 J mol(-1) K(-1)) and more negative DeltaC(p) (by about -75 J mol(-1) K(-1)) per water molecule. More generally, it is clear that significant DeltaC(p) effects are to be expected for any macromolecular process involving a multiplicity of cooperative weak interactions of whatever kind.     

Influence of substituent modifications on the binding of 2-amino-1,8-naphthyridines to cytosine opposite an AP site in DNA duplexes: thermodynamic characterization

Here, we report on a significant effect of substitutions on the binding affinity of a series of 2-amino-1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine (AND), 2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), all of which can bind to cytosine opposite an AP site in DNA duplexes. Fluorescence titration experiments show that the binding affinity for cytosine is effectively enhanced by the introduction of methyl groups to the naphthyridine ring, and the 1:1 binding constant (10⁶ M⁻¹) follows in the order of AND (0.30) < AMND (2.7) < ADMND (6.1) < ATMND (19) in solutions containing 110 mM Na⁺ (pH 7.0, at 20°C). The thermodynamic parameters obtained by isothermal titration calorimetry experiments indicate that the introduction of methyl groups effectively reduces the loss of binding entropy, which is indeed responsible for the increase in the binding affinity. The heat capacity change (ΔC_p), as determined from temperature dependence of the binding enthalpy, is found to be significantly different between AND (−161 cal/mol K) and ATMND (−217 cal/mol K). The hydrophobic contribution appears to be a key force to explain the observed effect of substitutions on the binding affinity when the observed binding free energy (ΔG_obs) is dissected into its component terms.     

Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta1,4-galactosyltransferase-I.

The lactose synthase (LS) enzyme is a 1:1 complex of a catalytic component, beta1,4-galactosyltransferse (beta4Gal-T1) and a regulatory component, alpha-lactalbumin (LA), a mammary gland-specific protein. LA promotes the binding of glucose (Glc) to beta4Gal-T1, thereby altering its sugar acceptor specificity from N-acetylglucosamine (GlcNAc) to glucose, which enables LS to synthesize lactose, the major carbohydrate component of milk. The crystal structures of LS bound with various substrates were solved at 2 A resolution. These structures reveal that upon substrate binding to beta4Gal-T1, a large conformational change occurs in the region comprising residues 345 to 365. This repositions His347 in such a way that it can participate in the coordination of a metal ion, and creates a sugar and LA-binding site. At the sugar-acceptor binding site, a hydrophobic N-acetyl group-binding pocket is found, formed by residues Arg359, Phe360 and Ile363. In the Glc-bound structure, this hydrophobic pocket is absent. For the binding of Glc to LS, a reorientation of the Arg359 side-chain occurs, which blocks the hydrophobic pocket and maximizes the interactions with the Glc molecule. Thus, the role of LA is to hold Glc by hydrogen bonding with the O-1 hydroxyl group in the acceptor-binding site on beta4Gal-T1, while the N-acetyl group-binding pocket in beta4Gal-T1 adjusts to maximize the interactions with the Glc molecule. This study provides details of a structural basis for the partially ordered kinetic mechanism proposed for lactose synthase.     

Thermodynamic characterization of ligand-induced conformational changes in UDP-N-acetylglucosamine enolpyruvyl transferase

The binding of UDP-N-acetylglucosamine (UDPNAG) to the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) was studied in the absence and presence of the antibiotic fosfomycin by isothermal titration calorimetry. Fosfomycin binds covalently to MurA in the presence of UDPNAG and also in its absence as demonstrated by MALDI mass spectrometry. The covalent attachment of fosfomycin affects the thermodynamic parameters of UDPNAG binding significantly: In the absence of fosfomycin the binding of UDPNAG is enthalpically driven (DeltaH = -35.5 kJ mol(-1) at 15 degrees C) and opposed by an unfavorable entropy change (DeltaS = -25 J mol(-1) K(-1)). In the presence of covalently attached fosfomycin the binding of UDPNAG is entropically driven (DeltaS = 187 J mol(-1)K(-1) at 15 degrees C) and associated with unfavorable changes in enthalpy (DeltaH = 28.8 kJ mol(-1)). Heat capacities for UDPNAG binding in the absence or presence of fosfomycin were -1.87 and -2.74 kJ mol(-1) K(-1), respectively, indicating that most ( approximately 70%) of the conformational changes take place upon formation of the UDPNAG-MurA binary complex. The major contribution to the heat capacity of ligand binding is thought to be due to changes in the solvent-accessible surface area. However, associated conformational changes, if any, also contribute to the experimentally measured magnitude of the heat capacity. The changes in solvent-accessible surface area were calculated from available 3D structures, yielding a DeltaC(p) of -1.3 kJ mol(-1) K(-1); i.e., the experimentally determined heat capacity exceeds the calculated one. This implies that other thermodynamic factors exert a large influence on the heat capacity of protein-ligand interactions.     

Val22对恶臭假单胞菌扁桃酸消旋酶催化活性的影响

恶臭假单胞菌扁桃酸消旋酶的Val22位于20 s环状结构上, 是与底物结合相关的氨基酸之一。其中Val被替换为Arg后酶活性下降了75.9%。除了酶与底物疏水作用减弱以外, 静电排斥作用增强也可能引起活性的下降。利用分子动力学模拟对酶与底物的米氏复合物进行分析, 结果表明: 突变后第22位氨基酸侧链与底物的静电势从0.036 kJ/mol升高至0.124 kJ/mol。这说明氨基酸侧链极性的改变增加了侧链与底物分子之间的静电排斥作用, 因而静电排斥作用也是导致突变体活性下降的原因之一。同时, 突变后系统势能增加了283 kJ/mol, 进一步证实了第22位氨基酸侧链极性和带电性质的改变导致酶与底物结合状态的势能增大, 从而引起活性大幅下降。因此, 将来对酶的结合口袋区域进行理性设计时, 除了考虑空间位阻效应外, 还需考虑疏水作用和静电作用。     

Xylose reductase from the Basidiomycete fungus Cryptococcus flavus: purification,steady-state kinetic characterization,and detailed analysis of the substrate binding pocket using structure-activity relationships

Xylose reductase has been purified to apparent homogeneity from cell extracts of the fungus Cryptococcus flavus grown on D-xylose as carbon source. The enzyme, the first of its kind from the phylum Basidiomycota, is a functional dimer composed of identical subunits of 35.3 kDa mass and requires NADP(H) for activity. Steady-state kinetic parameters for the reaction, D-xylose + NADPH + H(+)<--> xylitol + NADP(+), have been obtained at pH 7.0 and 25 degrees C. The catalytic efficiency for reduction of D-xylose is 150 times that for oxidation of xylitol. This and the 3-fold tighter binding of NADPH than NADP(+) indicate that the enzyme is primed for unidirectional metabolic function in microbial physiology. Kinetic analysis of enzymic reduction of aldehyde substrates differing in hydrophobic and hydrogen bonding capabilities with binary enzyme-NADPH complex has been used to characterize the substrate-binding pocket of xylose reductase. Total transition state stabilization energy derived from bonding with non-reacting sugar hydroxyls is approximately 15 kJ/mol, with a major contribution of 5-8 kJ/mol made by interactions with the C-2(R) hydroxy group. The aldehyde binding site is approximately 1.2 times more hydrophobic than n-octanol and can accommodate linear alkyl chains of 相似文献   

Measurement of thermodynamic parameters for hydrophobic mismatch 2: intermembrane transfer of a transmembrane helix

Hydrophobic matching between proteins and lipids is essential for the thermodynamic stability of integral membrane proteins. However, there is no direct thermodynamic information available about the intermembrane transfer of proteins between membranes with different hydrophobic thicknesses, which is crucial for understanding hydrophobic mismatch. This article reports the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the intermembrane transfer of the inert hydrophobic model transmembrane helix NBD-(AALALAA)(3)-NH(2) (NBD: 7-nitro-2-1,3-benzoxadiazol-4-yl), which is exchangeable between vesicles, from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to dimonounsaturated-phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 37-58 degrees C. The transfer free energies were calculated from equilibrium values of the extent of helix transfer from donor to acceptor lipid vesicles, as monitored by a decrease in fluorescence resonance energy transfer from the NBD group to a lipid-labeled Rhodamine in the donor upon transfer to the quencher-free acceptor. Under hydrophobic mismatch conditions up to approximately 7 A, the helix partitioning became unfavorable up to +7 kJ mol(-)(1), hampered by an increase in entropic (up to +20 kJ mol(-)(1)) and enthalpic (up to +66 kJ mol(-)(1)) terms in thinner and thicker membranes, respectively. Together with the results that H/D exchange at the membrane interface was accelerated in thinner membranes the obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, resulting in excess energies originating from the hydration of terminal hydrophobic residues or the unfavorable Born energy of terminal partial charges of the helix macrodipole.