Comparative investigations of biopolymer hydration by physicochemical and modeling techniques.

The comparative investigation of biopolymer hydration by physicochemical techniques, particularly by small-angle X-ray scattering, has shown that the values obtained differ over a wide range, depending on the nature of the polymer and the environmental conditions. In the case of simple proteins, a large number of available data allow the derivation of a realistic average value for the hydration (0.35 g of water per gram of protein). As long as the average properties of proteins are considered, the use of such a default value is sufficient. Modeling approaches may be used advantageously, in order to differentiate between different assumptions and hydration contributions, and to correctly predict hydrodynamic properties of biopolymers on the basis of their three-dimensional structure. Problems of major concern are the positioning and the properties of the water molecules on the biopolymer surface. In this context, different approaches for calculating the molecular volume and surface of biopolymers have been applied, in addition to the development of appropriate hydration algorithms.

A mutation affecting the association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri and its influence on the enzyme's activity and thermostability.

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunit of molecular mass 32 kDa. The enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. Oligomerization is required for enzyme activity and thermostability. We report here on a subunit interface mutation (R261E) which affects the dimer/tetramer part of the association equilibrium of formyltransferase. With the mutant protein it was shown that tetramerization is not required for activity but is necessary for high thermostability.

X-ray induced inactivation of the sulfhydryl enzyme malate synthase in the presence of various additives. Probing the extent of primary and post-irradiation inactivation and repair by rapid screening on the microlevel.

The sulfhydryl enzyme malate synthase was inactivated by X-irradiation in air-saturated aqueous solution, in the absence or presence of a variety of additives (thiols, antioxienzymes, typical radical scavengers, inorganic salts, buffer components, substrates, products, analogues). Radiation-induced changes of enzymic activity were registered immediately after stop of irradiation and in the post-irradiation period. Repair experiments were initiated by post-irradiation addition of dithiothreitol. Additionally, post-irradiation inactivation was modulated by some further additives. Probing the extent of primary and post-irradiation inactivation and repair was accomplished effectively by screening experiments on the microlevel, and by derivation of normalized efficiency parameters which allowed quick comparisons of the various additives with respect to their protective and repair-promotive efficiencies. Correlations between the efficiency parameters were studied by means of binary and ternary diagrams. Most of the substances added before irradiation were found to protect the enzyme against primary and post-irradiation inactivation and to increase the reparability of the enzyme by dithiothreitol, the extent of the effects depending on the nature (and concentration) of the additives used. Our results indicate that both specific protection (by substrates, products, analogues, and by sulfhydryl agents) and scavenging are responsible for the radioprotective efficiencies of the additives.

Primary and post-irradiation inactivation of the sulfhydryl enzyme malate synthase: correlation of protective effects of additives.

The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and post-irradiation inactivation. Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues). The results show that scavenging and specific protection are responsible for the protective efficiency of additives. Scavengers delete noxious species formed during irradiation or post-radiationem. Sulfhydryl reagents may act as repair substances. Specific ligands protect the active site of the enzyme and the essential sulfhydryls; specific protection is more pronounced post-radiationem. Ligands and sulfhydryl reagents may additionally act as scavengers. A cumulative index for the protective power of additives against both sorts of inactivation was established.

A small-angle X-ray scattering study on pre-irradiated malate synthase. The influence of formate, superoxide dismutase, and catalase on the X-ray induced aggregation of the enzyme.

The sulfhydryl enzyme malate synthase from baker's yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: congruent to 10 mg/ml; volume: 120 microliters), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase. After X-irradiation, a small aliquot of the irradiated solutions was tested for enzymic activity while the main portion was investigated by means of small-angle X-ray scattering. Additionally, an unirradiated sample without additives was investigated as a reference. Experiments yielded the following results: X-irradiation in the absence of the mentioned scavengers caused considerable aggregation, fragmentation, and inactivation of the enzyme. The dose Dt37 for total (= repairable + non-repairable) inactivation resulted as 4.4 kGy. The mean radius of gyration was found to be about 13 nm. The mean degree of aggregation was obtained as 5.7, without correction for fragmentation. An estimation based on the thickness factor revealed that about 19% of material might be strongly fragmented. When this amount of fragments was accordingly taken into account, a value of 7.1 was obtained as an upper limit for the mean degree of aggregation. The observed retention of the thickness factor and the finding of two different cross-section factors are in full accord with the two-dimensional aggregation model established previously (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980)). The presence of catalytic amounts of superoxide dismutase and/or catalase, in the absence of formate, during X-irradiation reduced both aggregation and inactivation significantly. The presence of formate (10 or 100 mM) during X-irradiation led to a strong decrease of aggregation and inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase. Effects of formate, superoxide dismutase, catalase, and dithiothreitol.

Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0, 30, 60 h after irradiation. To elucidate the role of OH., O-.2, and H2O2 in the X-ray inactivation of the enzyme, experiments were performed in the absence or presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Ar = 3% (corresponding to a D37 = 0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t = 0 led to Ar = 21%; repairs started later on resulted in somewhat lower activities. The decay of repairability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Ar = 72% and D37 = 6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. Both the presence of specific additives during irradiation and the addition of additives after irradiation may alter the post-irradiation inactivation. Catalase turned out to be the most potent inhibitor of post-irradiation inactivation; superoxide dismutase showed an ambivalent behaviour, it accelerated or impeded post-irradiation inactivation; formate, when added after irradiation, exhibited a moderate protective effect. The presence of specific additives, added before and/or after irradiation, influenced the repair behaviour to some extent. The highest activity achieved by repair amounted to about 90% of the activity of the corresponding unirradiated sample. The percentual gain of activity was found to be the greater the lower the residual activity of the enzyme was before initiation of repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Post-irradiation inactivation of the sulfhydryl enzyme malate synthase.

The sulfhydryl enzyme malate synthase was inactivated in air-saturated aqueous solution by X-irradiation (2 kGy). Changes of activity were registered up to 60 h after irradiation. Effects of specific additives (formate, superoxide dismutase, catalase), added before and/or after irradiation, revealed the role of the deleterious radical and non-radical species responsible for the radiation damage: inactivation during irradiation is mainly due to the action of OH, to a minor extent to O(2) and H2O2; post-irradiation inactivation is mainly caused by H2O2. A partial restoration of enzyme activity by dithiothreitol, added after irradiation, was found for all systems investigated; repairs were significant even when they were initiated 60 h after irradiation.

Electrophoretic and chemical studies on the X-ray damage of malate synthase.

The sulfhydryl enzyme malate synthase was shown to undergo an X-ray induced aggregation and inactivation in solution (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980). Further evidence for the occurrence of aggregation and inactivation and also of fragmentation and partial unfolding of the enzyme upon X-irradiation was obtained by chemical and electrophoretic studies. Irradiation was carried out in a specially designed microcell, experiments were performed on the microlevel. Under conditions of the experiments the formation of H2O2 upon X-irradiation could be proven; therefore the influence of H2O2 on the enzyme was investigated too. Though the quantitative results of the damaged enzyme particles are influenced by many disturbing factors, the findings allow clear statements on the nature of the effects under investigation. 1) Both X-irradiation and treatment with H2O2 caused a decrease of total and an increase of available sulfhydryl groups of the enzyme and led to a loss of enzymic activity. The presence of dithiothreitol turned out to be able to protect the enzyme against X-ray or H2O2 induced inactivation. Moreover, addition of dithiothreitol after X-irradiation or H2O2 treatment allowed a considerable repair of enzymic activity. 2) Polyacrylamide gel disc electrophoreses of X-irradiated enzyme solutions, performed in the presence of sodium dodecyl sulfate, showed the occurrence of covalently cross-linked subunits (preferably dimers and trimers) and of various definite fragments. Electrophoreses in the absence of the denaturant indicated the occurrence of enzyme aggregation. The effects were more pronounced with increasing X-ray doses. The electrophoreses also clearly reflected a radioprotection by dithiothreitol against cross-linking, but not against fragmentation. Addition of excess of 2-mercaptoethanol or of dithiothreitol to the X-irradiated enzyme clearly demonstrated that part of the covalent cross-links were disulfide bridges; the aggregates themselves, however, were held together primarily by non-covalent bonds. Blocking of exposed enzyme sulfhydryls by means of Ellman's reagent prevented both covalent cross-linking and enzyme aggregation. 3) Similar electrophoretic patterns as found for the X-irradiated enzyme were obtained for the unirradiated enzyme after treatment with H2O2. The similarity of the sulfhydryls in the presence of H2O2, suggest an involvement of H2O2 in the radiation damage of the enzyme. It seems plausible that oxidation reactions are responsible for the effects caused by X-irradiation or H2O2 treatment.

Large-scale purification and some properties of malate synthase from baker's yeast.

1. Malate synthase from baker's yeast (5 kg) was purified 400--500-fold to homogeneity. About 50--200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell-free crude extracts. The enzyme, pI = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies. 2. Sedimentation and diffusion coefficients were determined: S 0 20,w = 8.26 +/- 0.05 S, D 0 20,w = 4.5 +/- 0.1 X 10(-7) cm2 s-1. The molecular weight of the synthase was found to be 175 000 +/- 10 000 and 180 000 +/- 10 000 by sedimentation/diffusion and by high-speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180 000 +/- 10 000. 3. The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66 000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass. 4. The binding of acetyl-coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism. 5. The intermolecular isotopic effect, kH:k2H = 1.4, was determined with acetyl-coenzyme A and [2H3]acetyl-coenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H, 14C]acetyl-coenzyme A is also presented.

Small-angle X-ray scattering studies on the X-ray aggregation of malate synthase. Computer simulations and models.

Malate synthase undergoes an X-ray induced aggregation which can be monitored in situ by small-angle X-ray scattering; the analysis of scattering curves, taken at subsequent stages of aggregation, has led to the establishment of a tentative model for an aggregation in two dimensions (Zipper and Durchschlag (1980) Rad. and Environm. Biophys., in press). This model was checked by comparison of appropriate theoretical curves with the experimental curves. The theoretical scattering curves for this comparison were obtained by weighted averaging over the scattering curves calculated for various species of hypothetical aggregates. Based on the approximation of the unaggregated enzyme particle by an oblate cylinder, the aggregates were assumed to be composed of 2, 3, 4 or 6 of such cylinders, associated side-by-side in one and later on in two linear rows. THe weight fractions of the species were chosen so, that an optimum fit of the experimental mean radii of gyration and mean degrees of aggregation was achieved. The distance distribution functions calculated for the model are very similar to the functions derived from the scattering experiment. Cross-section Guinier plots of the scattering curves of the model reveal the occurrence of one and later on of two pseudo cross-section factors similar to those observed in the experimental scattering curves. The pseudo thickness factor of the model of the unaggregated particle is found to be retained in the model curves for all stages of aggregation. From these results it can be concluded that the model for the aggregation process is essentially consistent with the scattering behaviour of the aggregating enzyme. Small differences between the theoretical and experimental curves may be explained by the idealizations of the model. The comparison of theoretical curves for alternative models, assuming aggregation in three dimensions, suggests that these models are unlikely, though small amounts of three-dimensional aggregates cannot be ruled out completely.

Small-angle X-ray scattering on malate synthase from baker's yeast. The native substrate-free enzyme and enzyme-substrate complexes.

Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique. Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined. As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage. These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system. 1. For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm. Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder. The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm. Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape. 2. Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme. These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves). Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness. These changes imply an increase of the axial ratio by 2.2--6.9%, i.e. substrate binding induces a decrease of anisometry. While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter. In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA.

X-ray small-angle studies of the pyruvate dehydrogenase core complex from Escherichia coli K-12. I. Overall structure of the core complex.

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex which can be obtained with a unique polypeptide chain composition, has been investigated in solution with the X-ray small-angle technique. The molecular mass of the core complex of 3.78-10(6) daltons verifies the ratio of polypeptide chains of 16:16:16 of the three enzyme components, pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, present in the complex. In connection with the values obtained for the radius of gyration (156.5A), volume (1.07(7) A3) and amount of solvent associated with the complex (1.03 g/g) a loose packing of subunits in the complex has to be assumed. The maximum diameter of the core complex of 433 A, as determined from the correlation function, corroborates the large extension of the complex. The comparison of experimental and theoretical scattering curves reveals a relatively isometric overall shape of the core complex.