Conformational changes affect binding and catalysis by ester-hydrolysing antibodies

D2.3, D2.4 and D2.5 are ester-hydrolysing antibodies raised against a phosphonate transition state analogue (TSA). All three antibody-TSA binding kinetics, as monitored by fluorescence quenching, indicate an "induced-fit" mechanism: fast bimolecular association followed by a unimolecular isomerisation (k=1-7 s-1). Isomerisation leads to a 30-170-fold increase in affinity towards the TSA and, consequently, to higher catalytic rates. Antibody D2.3 exhibits a complex three-step binding mechanism, in which the last step is a "very slow" isomerisation (k<0.02 s-1). This very slow isomerisation is limiting the rate of catalysis by D2.3, as indicated by the kinetics of product release which show characteristics of enzyme "conformational memory" or "hysteresis". The results support a mechanism consisting of pre-equilibrium between "nether-active" (low affinity) and "active" (high affinity) antibody conformers (prior to ligand addition) as well as induced-fit, i.e. isomerisation of the nether-active ligand-antibody complex to give the active complex. Crystal structures of these antibodies, free and complexed, have previously indicated that their conformation does not change upon binding. Here, we show that the buffer used to crystallise the antibodies, and in particular its polyethylene glycol component, alters the pre-equilibrium in favour of the active conformer, leading to its crystallisation both in the presence and in the absence of the TSA.     

Thymidylate synthase: structure, inhibition, and strained conformations during catalysis

Thymidylate synthase (TS) is a long-standing target for chemotherapeutic agents because of its central role in DNA synthesis, and it is also of interest because of its rich mechanistic features. The reaction catalyzed by TS is the methylation of dUMP, with the transferred methyl group provided by the cofactor methylenetetrahydrofolate (CH2THF). Recently, several crystal structure determinations and mechanistic studies have led to a deeper understanding of the TS reaction mechanism, and address the role of conformational change in TS catalysis and inhibition. Included among these structures are complexes of TS bound to substrate dUMP; cofactor CH2THF; the nucleotide analogs 5-fluoro-dUMP, 5-nitro-dUMP and dGMP; and the promising antifolates BW1843, ZD1694, and AG337. From these studies, a picture of TS emerges where ligand-induced conformational changes play key roles in catalysis by straining the thiol adduct that occurs during the reaction; by protecting the highly reactive reaction intermediates; and by providing a means to stabilize a high-energy conformer of the cofactor after initial binding of a low-energy conformer. The best inhibitors of TS also induce and stabilize a conformational change in TS. One inhibitor, BW1843, distorts the active site on binding, and intercalates into a hydrophobic patch between two mobile subdomains in the protein. Also discussed are recent developments in the cell biology and regulation of eukaryotic TS and the use of structure-based drug design in the development of the antifolates currently in clinical trial for the treatment of cancer.     

Age dependent changes in mitochondrial FoF1 ATP synthase in regenerating rat-liver

The effect of aging on rat liver regeneration and on the FoF1-ATP synthase complex of isolated liver mitochondria was followed after partial (70%) hepatectomy. ATP hydrolase activity in submitochondrial particles prepared from regenerating liver was first depressed; the time needed to reach the lowest activity was age dependent. This decrease was accompanied by parallel decrease of i) the respiratory rate of succinate supplemented mitochondria in state III; ii) the respiratory control index; iii) the rate of synthesis of ATP in succinate supplemented submitochondrial particles. This first phase of liver regeneration, characterized at all ages by a lag phase in the growth, was followed by a second phase in which the tissue mass was restored and the enzyme activities normalized. Immunoblot analysis showed that the changes in the catalytic activities of the FoF1-ATP synthase observed during liver regeneration were accompanied by parallel changes in the amount of subunits of both the catalytic (F1) and the membrane (Fo) sector of the complex.     

Mapping the transition state for ATP hydrolysis: implications for enzymatic catalysis

BACKGROUND: Phosphoryl transfer, typically involving high energy phosphate donors such as ATP, is the most common class of biological reactions. Despite this, the transition state for phosphoryl transfer from ATP in solution has not been systematically investigated. Characterization of the transition state for the uncatalyzed hydrolysis of ATP would provide a starting point for dissection of enzyme-catalyzed reactions. RESULTS: We examined phosphoryl transfer from ATP, GTP and pyrophosphate to a series of alcohols; these reactions are analogous to the phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. The Br?nsted beta(nucleophile) value of 0.07 is small, indicating that there is little bond formation between the incoming nucleophile and the electrophilic phosphoryl group in the transition state. Coordination of Mg2+ has no measurable effect on this value. The Br?nsted beta(leaving group) value of -1.1 for phosphoryl transfer to water from a series of phosphoanhydrides is large and negative, suggesting that the bond between phosphorous and the leaving group oxygen is largely broken in the transition state. CONCLUSIONS: Uncatalyzed hydrolysis of ATP in solution occurs via a dissociative, metaphosphate-like transition state, with little bond formation between nucleophile and ATP and substantial cleavage of the bond between the gamma-phosphoryl moiety and the ADP leaving group. Bound Mg2+ does not perturb the dissociative nature of the transition state, contrary to proposals that enzyme-bound metal ions alter this structure. The simplest expectation for phosphoryl transfer at the active site of enzymes thus entails a dissociative transition state. These results provide a basis for analyzing catalytic mechanisms for phosphoryl transfer.     

Energy transduction in ATP synthase

Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning F0 portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through F0 is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil gamma-subunit. This acts as a rotating 'cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this proton motive force biases the rotor's diffusion so that F0 constitutes a rotary motor turning the gamma shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility-supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump-can also be explained by our model.     

A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.     

Conformational changes of active sites during refolding of urea-denatured creatine kinase

The course of the recovery of the active site conformation during refolding of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. The recovery of the active site conformation has been followed by changes in probe fluorescence of active site of the enzyme labeled by o-phthaldehyde and has been shown to be a triphasic process. A comparison of the rate constants for the conformational recovery of the active site with those for the recovery of enzyme catalytic activity shows that these are synchronized. The results obtained show clearly that, although recovery of partial activity and conformation of the active site occur in reactions with nearly the same rate as those of the refolding processes observed, the complete recovery of the enzyme activity and the active site conformation can only be obtained long after any detectable conformational change of whole enzyme molecule.     

Conformational change rate-limits GTP hydrolysis: the mechanism of the ATP sulfurylase-GTPase

The fluorescent GTP analogues 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imidotriphosphate) (mGMPPNP) and 3'-O-(N-methylanthraniloyl)-2'-deoxy-GTP (mGTP) were used to demonstrate that an enzyme isomerization precedes and rate-limits beta,gamma-bond cleavage in the catalytic cycle of the ATP sulfurylase-GTPase, from E. coli K-12. The binding of mGMPPNP to the E.AMP.PPi complex of ATP sulfurylase is biphasic, indicating that an isomerization occurs in the binding reaction. The isomerization mechanism was assigned based on the results of the enzyme concentration dependence of the observed rate constants, kobs, for both phases of the binding reaction, and sequential-mixing, nucleotide release experiments. The isomerization occurs after, and is driven by, the addition of mGMPPNP. Values were determined for each of the rate constants associated with the two-step kinetic model used in the interpretation of the results. A comparison of the enzyme concentration dependence of kobs for the hydrolysis and binding reactions reveals that the rate constants for the corresponding steps of these two reactions are extremely similar. The virtually identical rate constants for isomerization and beta, gamma-bond scission strongly suggest that isomerization rate-limits bond breaking. The implications of these finding for GTPase/target interactions and the mechanism of energetic linkage in the ATP sulfurylase system are discussed.     

Isolation of supernumerary yeast ATP synthase subunits e and i. Characterization of subunit i and disruption of its structural gene ATP18

Two subunits of the yeast ATP synthase have been isolated. Subunit e was found loosely associated to the complex. Triton X-100 at a 1% concentration removed this subunit from the ATP synthase. The N-terminal sequencing of subunit i has been performed. The data are in agreement with the sequence of the predicted product of a DNA fragment of Saccharomyces cerevisiae chromosome XIII. The ATP18 gene encodes subunit i, which is 59 amino acids long and corresponds to a calculated mass of 6687 Da. Its pI is 9.73. It is an amphiphilic protein having a hydrophobic N-terminal part and a hydrophilic C-terminal part. It is not apparently related to any subunit described in other ATP synthases. The null mutant showed low growth on nonfermentable medium. Mutant mitochondria display a low ADP/O ratio and a decrease with time in proton pumping after ATP addition. Subunit i is associated with the complex; it is not a structural component of the enzyme but rather is involved in the oxidative phosphorylations. Similar amounts of ATP synthase were measured for wild-type and null mutant mitochondria. Because 2-fold less specific ATPase activity was measured for the null mutant than for the wild-type mitochondria, we make the hypothesis that the observed decrease in the turnover of the mutant enzyme could be linked to a proton translocation defect through F0.     

Turnover number of Escherichia coli F0F1 ATP synthase for ATP synthesis in membrane vesicles

The rate of ATP synthesized by the ATP synthase (F0F1-ATPase) is limited by the rate of energy production via the respiratory chain, when measured in everted membrane vesicles of an Escherichia coli atp wild-type strain. After energization of the membranes with NADH, fractional inactivation of F0F1 by the covalent inhibitor N,N'-dicyclohexylcarbodiimide allowed the rate of ATP synthesis/mol remaining active ATP synthase complexes to increase; the active ATP synthase complexes were calculated using ATP hydrolysis rates as the defining parameter. In addition, variation of the assay temperature revealed an increase of the ATP synthesis rate up to a temperature of 37 degrees C, the optimal growth temperature of E. coli. In parallel, the amount of F0F1 complexes present in membrane vesicles was determined by immunoquantitation to be 3.3 +/- 0.3% of the membrane protein for cells grown in rich medium and 6.6 +/- 0.3% for cells grown in minimal medium with glycerol as sole carbon and energy source. Based on these data, a turnover number for ATP synthesis of 270 +/- 40 s(-1) could be determined in the presence of 5% active F0F1 complexes. Therefore, these studies demonstrate that the ATP synthase complex of E. coli has, with respect to maximum rates, the same capacity as the corresponding enzymes of eukaryotic organells.     

PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.     

Conformational changes in conantokin-G induced upon binding of calcium and magnesium as revealed by NMR structural analysis

The apo- and metal-bound solution conformations of synthetic conantokin-G (con-G, G1Egamma gammaL5Q gamma NQgamma 10LIRgamma K15SN-CONH2, gamma = gamma-carboxyglutamic acid), an antagonist of N-methyl-D-aspartate receptor-derived neuronal ion channels, have been examined by one- and two-dimensional 1H NMR at neutral pH. A complete structure for the Mg2+-loaded peptide was defined by use of distance geometry calculations and was found to exist as an alpha-helix that spans the entire peptide. The alpha-helical nature of Mg2+/con-G was also supported by the small values (<5.5 Hz) of the 3JHNalpha coupling constants measured for amino acid residues 3-5, 8, 9, and 11-16, and the small values (<4 ppb/K) of the temperature coefficients observed for the alphaNH protons of residues 5-17. This conformation contrasted with that obtained for apo-con-G, which was nearly structureless in solution. Docking of Mg2+ into con-G was accomplished by use of the genetic algorithm/molecular dynamics simulation method, employing the NMR-derived Mg2+-loaded structure for initial coordinates in the midpoint calculations. For the 3 Mg2+/con-G model, it was found that binding of one Mg2+ ion is stabilized by oxygen atoms from three gamma-carboxylates of Gla3, Gla4, and Gla7; another Mg2+ is coordinated by two oxygen atoms, one from each of the gamma-carboxylates of Gla7; and a third metal ion through three donor oxygen atoms of gamma-carboxylates from Gla10 and Gla14. As shown from direct metal binding measurements to mutant con-G peptides, these latter two Gla residues probably stabilized the tightest binding Mg2+ ion. Circular dichroism studies of these same peptide variants demonstrated that all Gla residues contribute to the adoption of the Mg2+-dependent alpha-helical conformation in con-G. The data obtained in this investigation provide a molecular basis for the large conformational alteration observed in apo-con-G as a result of divalent cation binding and allow assessment of the roles of individual Gla residues in defining certain of the structure-function properties of con-G.     

Age-related changes in selective attention and perceptual load during visual search.

Three visual search experiments were conducted to test the hypothesis that age differences in selective attention vary as a function of perceptual load (E. A. Maylor & N. Lavie, 1998). Under resource-limited conditions (Experiments 1 and 2), the distraction from irrelevant display items generally decreased as display size (perceptual load) increased. This perceptual load effect was similar for younger and older adults, contrary to the findings of Maylor and Lavie. Distraction at low perceptual loads appeared to reflect both general and specific inhibitory mechanisms. Under more data-limited conditions (Experiment 3), an age-related decline in selective attention was evident, but the age difference was not attributable to capacity limitations as predicted by the perceptual load theory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Solid structural changes during the reduction of iron oxides

Abstract

The changes in the solid structure, i.e., grain size, shape and pore structure, during the reduction. of reagent-grade hematite to iron by CO?CO₂ mixtures at temperatures between 725 and 925°C were investigated. The reduction of hematite to wustite was found to produce little structural change. In contrast, three mechanisms of structural change occurred during the reduction of wustite to iron. These included the thermal sintering of the wustite, the growth of fibrous iron filaments from the partially reduced wustite grains, and the sintering or “coarsening” of the reduced iron. A quantitative model for the sintering of the wustite is described.

The effects of the structural changes on the reduction kinetics are reviewed. The sintering of the wustite reduced the over-all rate of reduction; the growth of the fibrous iron increased the rate of reduction. The sintering of the iron had little effect on the kinetics. The interaction between the sintering of the wustite and the growth of the fibrous irons, which change the character of the reduction from nontopochemical to topochemical, is also discussed.

Résumé

Les variations de la structure solide, i.e., la grosseur du grain, la forme et la porosité,ont été étudiées au moment de la réduction d'hématite en fer, dans des mélanges de CO?CO₂ aux températures comprises entre 725 et 925°C. La réduction de l'hématite à la wustite s'accompagne d'un léger changement de structure. Au contraire, trois mécanismes de changements structuraux se sont présentés au cours de la réduction de la wustite en fer. Ce sont: le frittage thermique de la wustite, la croissance des filaments fibreux de fer provenant des grains de wustite partiellement réduits et aussi le frittage du fer réduit. Un modéle quantitatif décrit Ie frittage de la wustite.

Les effets des variations structurales sur la cinétique de réduction sont revisés. Le frittage de la wustite a diminué le taux de réduction, alors que la croissancé du fer fibreux a augmente ce taux. Le frittage du fer a un effet minime sur la cinétique. L'interaction entre le frittage de la wustite et la croissance des fibres de fer fait varier Ie caractère de la réduction, de non-topochimique à topochimique.     

Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis

The involvement of the protoporphyrin IX heme iron of macrophage nitric oxide synthase (NOS) in the oxidation of NG-hydroxy-L-arginine (L-NHA) to nitric oxide (NO) and citrulline was investigated by carbon monoxide (CO) inhibition studies and binding difference spectroscopy. A CO:oxygen mixture (80:20) was found to inhibit the reaction by 33% with L-NHA as a substrate compared to 57% with L-arginine. Spectral perturbations were observed upon the addition of L-NHA to oxidized NOS, producing a type I binding difference spectrum with a maximum at 384 nm and minimum at 420 nm. In addition, L-NHA was incapable of reducing anaerobic oxidized NOS in the absence of NADPH. These studies support the involvement of the heme in the oxidation of L-NHA to NO and citrulline, indicating that the heme functions in both of the currently characterized oxidative steps of the NOS reaction.     

Tyrosine structural changes detected during the photoactivation of rhodopsin

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho --> MII difference spectra were obtained at 10 degrees C for unlabeled recombinant Rho and isotope-labeled L-[ring-2H4]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2H4]Tyr and isolated using a His6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR --> M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the F-helix, may form a hinge that facilitates alpha-helix movement.     

Dexamethasone-induced changes in endometrial growth and inducible nitric oxide synthase during decidualization in rats

1. The present study investigated the time-dependent inhibitory responses of endometrial growth and inducible nitric oxide synthase (iNOS) to dexamethasone during deciduoma development that was surgically induced on day 4 of pseudopregnancy (PG). 2. Groups of rats (n = 6) were subcutaneously injected with dexamethasone (1.5 mg/rat per day) for 3 days (PG days 1-3, 4-6, 7-9, 10-12 and 12-15). Rats in each group were killed on the last injection day. 3. Dexamethasone produced comparable temporal inhibitory changes in endometrial growth (wet weight, protein and DNA concentrations; P<0.0001) and in iNOS activity (130 kDa protein band), which peaked after PG days 4-6 and 7-9 pretreatments. 4. Endometrial matrix metalloproteinases (72 and 92 kDa) activity profiles displayed maximal reductions (36 and 53%, respectively) following PG days 4-6 pretreatment. Serum progesterone levels were equally (P<0.0001) but asynchronously inhibited by dexamethasone on PG days 9 and 12. 5. Dexamethasone inhibition of endometrial growth and in situ iNOS was most pronounced during decidual development (PG days 4-9). Minor reductions in these endometrial parameters occurred before deciduoma induction (PG days 1-3) and during deciduoma regression (PG days 10-15). 6. These results indicate that, in the endometrium, the iNOS/endogenous nitric oxide system may be linked to the biochemical and metabolic mechanisms responsible for the developmental responsiveness of the deciduoma to dexamethasone exposure. These time-dependent changes in endometrial growth and iNOS apparently were not mediated by progesterone.