Diethylbarbiturate potentiation of 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation

The rate of rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate is increased in the presence of diethylbarbiturate in the reaction medium. A "rate saturation effect" indicates the formation of a rhodanese-diethylbarbiturate complex, prior to modification-induced enzyme inactivation. The dissociation constant of this complex is 19.0 mM. Diethylbarbiturate has no effect on the trinitrophenylation rate of the free amino groups of rhodanese. When rhodanese modification, in the presence of diethylbarbiturate in the reaction medium, is carried out by the use of a 2,4,6-trinitrobenzenesulphonate concentration much lower than the concentration of rhodanese modifiable amino groups, reaction stoichiometry indicates that 3 to 5 moles of rhodanese are rendered inactive for each mole of 2,4,6-trinitrobenzenesulphonate utilized. This finding indicates the existence of a chain-reaction type mechanism of rhodanese inactivation.     

Modification and inactivation of rhodanese by 2,4,6-trinitrobenzenesulphonic acid

Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.     

Kinetics of protein-modification reactions. Stoichiometry of modification-produced enzyme inactivation: modification of rhodanese by 2,4,6-trinitrobenzenesulphonic acid.

A mathematical treatment is presented for the dependence of enzyme activity loss on the numbers and reactivities of the groups essential for catalytic function, when enzyme protein modification is carried out by the use of concentrations of protein reactive groups well in excess of that of modifying agent. Experimentally obtained data on the modification of rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) by 2,4,6-trinitrobenzenesulphonic acid are presented, and it is shown that, at pH9.00, the fractional concentration of rhodanese groups, or of rhodanese group reactivities, essential for enzyme catalytic function is 0.88; this value is found to decrease with decreasing pH of the reaction medium. The possibility that rhodanese inactivation by 2,4,6-trinitrobenzenesulphonic acid is brought about by modification of groups other than amino groups is ruled out by a comparison of the enzyme-inactivation and protein-modification stoichiometries, for putative reaction models for enzyme and modifying agent.     

Oxidative inactivation of the enzyme rhodanese by reduced nicotinamide adenine dinucleotide

The enzyme rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is inactivated with a half-time of approximately 3 min when incubated with 50 mM NADH. NAD+, however, has virtually no effect on the activity. Inactivation can be prevented by the inclusion of the substrate thiosulfate. The concentration of thiosulfate giving half-protection is 0.038 mM. In addition, NADH, but not NAD+, is a competitive inhibitor with respect to thiosulfate in the catalyzed reaction (Ki = 8.3 mM). Fluorescence studies are consistent with a time-dependent oxidation of NADH in the presence of rhodanese. The sulfur-free form of rhodanese is more rapidly inactivated than the sulfur-containing form. Spectrophotometric titrations show that inactivation is accompanied by the loss of two free SH groups per enzyme molecule. Inactivation is prevented by the exclusion of air and the inclusion of EDTA (1 mM), and the enzyme activity can be largely protected by incubation with superoxide dismutase or catalase. Rhodanese, inactivated with NADH, can be reactivated by incubation with the substrate thiosulfate (75 mM) for 48 h or more rapidly, but only partially, by incubating with 180 mM dithiothreitol. It is concluded that, in the presence of rhodanese, NADH can be oxidized by molecular oxygen and produce intermediates of oxygen reduction, such as superoxide and/or hydrogen peroxide, that can inactivate the enzyme with consequent formation of an intraprotein disulfide. In addition, NADH, but not NAD+, can reversibly bind to the active site region in competition with thiosulfate. These data are of interest in view of x-ray studies that show structural similarities between rhodanese and nucleotide binding proteins.     

Acceptor substrate-potentiated inactivation of bovine liver rhodanese

The interaction of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) with the acceptor substrates, dithiothreitol or cyanide, was studied. When incubated in the presence of cyanide or dithiothreitol, rhodanese was inactivated in a time-dependent process. This inactivation was detectable only at low enzyme concentrations; the rate and degree of inactivation could be modulated by varying the substrate concentration or the system pH. Activity measurements and fluorescence spectroscopy techniques were used in examining the inactivation phenomenon. Sulfur transfer to dithiothreitol was measured by direct assay and was shown to involve the dequenching of enzymic intrinsic fluorescence that had been previously observed only with cyanide as the acceptor substrate. Substrate-potentiated inactivation of rhodanese (with cyanide) has been reported before, but the cause and nature of this interaction were unexplained. The results presented here are consistent with an explanation invoking oxidation of rhodanese in the course of inactivation.     

Interaction of rhodanese with intermediates of oxygen reduction

Cyanide-promoted inactivation of the enzyme rhodanese [thiosulfate sulfurtransferase (EC 2.8.1.1)] in the presence of ketoaldehydes is caused by reduced forms of molecular oxygen generated during autoxidation of the reaction products. The requirement of both catalase and superoxide dismutase to prevent rhodanese inactivation indicates that hydroxyl radical could be the most efficient inactivating agent. Rhodanese, also in the less stable sulfur-free form, shows a different sensitivity towards oxygen activated species. While the enzyme is unaffected by superoxide radical, it is rapidly inactivated by hydrogen peroxide. The extent of inactivation depends on the molar ratio between sulfur-free enzyme and oxidizing agent. Fully inactive enzyme is reactivated by reduction with its substrate thiosulfate.     

Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures.

The chaperonin protein cpn60 from Escherichia coli protects the monomeric, mitochondrial enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) against heat inactivation. The thermal inactivation of rhodanese was studied for four different states of the enzyme: native, refolded, bound to cpn60 in the form of a binary complex formed from unfolded rhodanese, and a thermally perturbed state. Thermal stabilization is observed in a range of temperatures from 25 to 48 degrees C. Rhodanese that had been inactivated by incubation at 48 degrees C, in the presence of cpn60 can be reactivated at 25 degrees C, upon addition of cpn10, K+, and MgATP. A recovery of about 80% was achieved after 1 h of the addition of those components. Thus, the enzyme is protected against heat inactivation and kept in a reactivable form if inactivation is attempted using the binary complex formed between rhodanese folding intermediate(s) and cpn60. The chaperonin-assisted refolding of urea-denatured rhodanese is dependent on the temperature of the refolding reaction. However, optimal chaperonin assisted refolding of rhodanese observed at 25 degrees C, which is achieved upon addition of cpn10 and ATP to the cpn60-rhodanese complex, is independent of the temperature of preincubation of the complex, that was formed previously at low temperature. The results are in agreement with a model in which the chaperonin cpn60 interacts with partly folded intermediates by forming a binary complex which is stable to elevated temperatures. In addition, it appears that native rhodanese can be thermally perturbed to produce a state different from that achieved by denaturation that can interact with cpn60.     

Inactivation of rhodanese by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate.     

Chemical modification of bovine liver rhodanese with tetrathionate: differential effects on the sulfur-free and sulfur-containing catalytic intermediates

Sulfhydryl groups of bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) were modified by treatment with tetrathionate. There was a linear relationship between loss of enzyme activity and the amount of tetrathionate used. At a ratio of one tetrathionate per mole of rhodanese, 100% of enzyme activity was lost in the sulfur-free E-form as compared with a 70% loss for the sulfur-containing ES-form of the enzyme. Addition of up to a 100-fold molar excess of tetrathionate to ES gave no further inactivation. Addition of cyanide to the maximally inactivated ES-tetrathionate complex gave complete loss of activity. Kinetic studies of maximally inactivated ES and partially inactivated E gave Km (Ks) values that were essentially the same as native enzyme, indicating that the active enzyme, in all cases, bound thiosulfate similarly. Reactivation was faster with the ES-form than with the E-form. The substrate, thiosulfate, could reactivate the enzyme up to 70% in 1 h with ES as compared to 24 h with E. Tetrathionate modification of rhodanese could be correlated with the changes in intrinsic fluorescence and with the binding of the active site reporter 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS). Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. Cadmium chloride and phenylarsine oxide totally inactivated the enzyme at levels usually associated with their effect on enzymes containing vicinal sulfhydryl groups. Further, cadmium inhibition could be reversed by EDTA. Tetrathionate modification of rhodanese may proceed through the formation of sulfenylthiosulfate intermediates at sulfhydryl groups, close to but not identical with the active-site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges.     

Chemical Modification of Rhodanese with Sulphite

The essential sulphydryl group of bovine liver rhodanese (thiosulphate: cyanide sulphurtrasferase, E.C. 2.8.1.1.) is modified by sulphite produced during the enzymatic reaction or added to the fully active enzyme. The enzyme treated with labelled reagent incorporates 1 equivalent of SO²₃^- and loses one -SH group with the formation of a S-sulphonate group at the active site. Mercaptoethanol is effective in both restoring enzyme activity and removing bound sulphite from protein. The inactivation process is dependent on the presence of oxygen and is antagonized by chelation of metal ions, that catalyze sulphite autoxidation, or by scavenging free radicals with mannitol or benzoate. Since the presence of superoxide dismutase and/or catalase protects the enzyme only to a small extent, the inactivation process should be attributed to sulphite radicals rather than intermediates of oxygen reduction.     

Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation

Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.     

Chemical modification of bovine liver rhodanese with tetrathionate: differential effects or the sulfur-free and sulfur-containing catalytic intermediates

Sulfhydryl groups of bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) were modified by treatment with tetrathionate. There was a linear relationship between loss of enzyme activity and the amount of tetrathionate used. At a ratio of one tetrathionate per mole of rhodanese, 100% of enzyme activity was lost in the sulfur-free E-form as compared with a 70% loss for the sulfur-containing ES-form of the enzyme. Addition of up to a 100-fold molar excess of tetrathionate to ES gave no further inactivation. Addition of cyanide to the maximally inactivated ES-tetrathionate complex gave complete loss of activity. Kinetic studies of maximally inactivated ES and partially inactivated E gave K_m (K₅) values that were essentially the same as native enzyme, indicating that the active enzyme, in all cases, bound thiosulfate-similarly. Reactivation was faster with the ES-form than with the E-form. The substrate, thiosulfate, could reactivate the enzyme up to 70% in 1 h with ES as compared to 24 h with E. Tetrathionate modification of rhodanese could be correlated with the changes in intrinsic fluorescence and with the binding of the active site reporter 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS). Circular dichroism spectra of the protein suggested increased ordered secondary structure in the protein after reaction with tetrathionate. Cadmium chloride and phenylarsine oxide totally inactivated the enzyme at levels usually associated with their effect on enzymes containing vicinal sulfhydryl groups. Further, cadmium inhibition could be reserved by EDTA. Tetrathionate modification of rhodanese may proceed through the formation of sulfenylthiosulfate intermediates at sulfhydryl groups, close to but not identical with the active-site sulfhydryl group, which then can react further with the active-site sulfhydryl group to form disulfide bridges.     

alpha-Crystallin facilitates the reactivation of hydrogen peroxide-inactivated rhodanese

It was previously shown that rhodanese, inactivated with hydrogen peroxide, could only be reactivated in the presence of a reductant or the substrate thiosulfate if these reagents were added soon after inactivation and if the oxidant was removed. Here, we report on the facilitated reactivation (75%) of hydrogen peroxide-inactivated rhodanese by the chaperone alpha-crystallin. Reactivation by the chaperone still required a reductant and thiosulfate. Without alpha-crystallin, but in the presence of the reductant and thiosulfate, the inactivated enzyme regained about 39% of its original activity. The alpha-crystallin-assisted reactivation of hydrogen peroxide-inactivated rhodanese was independent of ATP. Further, we found, that alpha-crystallin interacted transiently, but could not form a stable complex with hydrogen peroxide-inactivated rhodanese. Unlike in prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that alpha-crystallin can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein.     

Reversible thermal denaturation of immobilized rhodanese

For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [35S]SO3(2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states.     

Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function.

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5'-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.     

Production of rhodanese by bacteria present in bio-oxidation plants used to recover gold from arsenopyrite concentrates

Considerably larger quantities of cyanide are required to solubilize gold following the bio-oxidation of gold-bearing ores compared with oxidation by physical-chemical processes. A possible cause of this excessive cyanide consumption is the presence of the enzyme rhodanese. Rhodanese activities were determined for the bacteria most commonly encountered in bio-oxidation tanks. Activities of between 6.4 and 8.2 micromol SCN min(-1) mg protein(-1) were obtained for crude enzyme extracts of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Thiobacillus caldus, but no rhodanese activity was detected in Leptospirillum ferrooxidans. Rhodanese activities 2-2.5-fold higher were found in the total mixed cell mass from a bio-oxidation plant. T. ferrooxidans synthesized rhodanese irrespective of whether it was grown on iron or sulphur. With a PCR-based detection technique, only L. ferrooxidans and T. caldus cells were detected in the bio-oxidation tanks. As no rhodanese activity was associated with L. ferrooxidans, it was concluded that T. caldus was responsible for all of the rhodanese activity. Production of rhodanese by T. caldus in batch culture was growth phase-dependent and highest during early stationary phase. Although the sulphur-oxidizing bacteria were clearly able to convert cyanide to thiocyanate, it is unlikely that this rhodanese activity is responsible for the excessive cyanide wastage at the high pH values associated with the gold solubilization process.     

Chemical modification of glutamate dehydrogenase by 2,4,6-trinitrobenzenesulphonic acid

1. Modification with 2,4,6-trinitrobenzenesulphonic acid was studied for its effect on the structure, activity and response to regulatory effectors of ox liver glutamate dehydrogenase. 2. The modification affected amino groups only, and the relative reactivities of the amino groups of the enzyme are described. 3. A biphasic inactivation of the enzyme was observed and analysis of the course of inactivation and of modification showed that the rapid reaction of one amino group/subunit leads to loss of 80% of the enzymic activity. 4. NADH retarded the inactivation by 2,4,6-trinitrobenzenesulphonic acid, the protection increasing with NADH concentration. This, together with the previous observation, suggests that the rapidly reacting group is essential for the activity of the enzyme. 5. The effects of modification on the optical-rotatory-dispersion and sedimentation behaviour of the enzyme were studied. 6. The enzyme's response to the allosteric effector GTP was rapidly lost on modification, whereas its response to ADP was unaffected. Comparison of the inactivation and desensitization suggests that the reactive amino group is essential for both activity and GTP response, and that only a completely unmodified enzyme oligomer responds fully to GTP. 7. The merits of chemical-modification studies of large enzymes are discussed critically in connexion with the interpretation of these results.     

Inhibition of the catalytic activity of rhodanese by S-nitrosylation using nitric oxide donors

Rhodanese (EC 2.8.1.1.) from bovine liver contains four reduced cysteine groups. The –SH group of cysteine 247, located in a rhodanese active centre, transfers sulfane sulfur in a form of hydrosulfide (–S–SH) from appropriate donors to nucleophilic acceptors. We aimed to discover whether S-nitrosylation of critical cysteine groups in rhodanese can inhibit activity of the enzyme by covalent modification of –SH groups.

The inhibition of rhodanese activity was studied with the use of a number of nitric oxide (NO) donors. We have successfully confirmed using several methods that the inhibition of rhodanese activity is a result of the formation of stable S-nitrosorhodanese.

Low molecular weight NO donors, such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), inactivate rhodanese and are much more effective in this regard (100% inhibition at 2.5 mM) than such known inhibitors of this enzyme, as N-ethylmaleimide (NEM) (25 mM < 50%) or sulfates(IV) (90% inhibition at 5 mM). On the other hand, sodium nitroprusside (SNP) and nitrites inhibit rhodanese activity only in the presence of thiols, which suggests that S-nitrosothiols (RSNO) also have to participate in this reaction in this case.

A demonstration that rhodanese activity can be inhibited as a result of S-nitrosylation suggests the possible mechanism by which nitric oxide may regulate sulfane sulfur transport to different acceptors.     

Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound,inactivated, and reactivated by GroEL

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.     

Occurrence of the thiosulphate sulphurtransferase producers in the population of mesophilic heterotrophic bacteria and microfungi of spruce humus

The activity of thiosulphate sulphurtransferase (rhodanese, EC 2.8.1.1) in randomly isolated bacteria and mieromycetes of the humus horizon in a spruce forest was followed. Bacteria isolated on soil extract agar (70 cultures) did not yield unambiguous results due to poor growth of the cultures. Of 63 bacterial cultures on meat-peptone agar almost 80 % of the isolates produced the enzyme. Sixty-three % of isolates had a concentration higher than 5 nkat/L and 35 % higher than 20 nkat/L. The soil rhodanese activity can be explained by their presence. None of 30 isolates of micromycetes produced thiocyanate.