Sulfhemoglobin formation in human erythrocytes by cystalysin, an L-cysteine desulfhydrase from Treponema denticola.

Cystalysin, isolated from the oral pathogen Treponema denticola, is an L-cysteine desulfhydrase (producing pyruvate, ammonia and hydrogen sulfide from cysteine) that can modify hemoglobin and has hemolytic activity. Here, we show that enzymatic activity of recombinant cystalysin depends upon stochiometric pyridoxal phosphate. The enzyme was not functional as an L-alanine transaminase, and had a strong preference for L-cysteine over D-cysteine. Cystalysin preferred small alpha-L-amino acids as substrates or inhibitors and was far more active towards L-cysteine than towards the other standard amino acids that undergo pyridoxal phosphate-dependent beta-elimination reactions (serine, threonine, tryptophan and tyrosine). Cystalysin tolerated small modifications to the carboxylate of L-cysteine (i.e., the methyl and ethyl esters of L-cysteine were good substrates), but the smallest possible peptide with an N-terminal cysteine, L-cysteinylglycine, was a very poor substrate. These results, combined with the implicit requirement for a free amine for pyridoxal phosphate-dependent reactions, imply that cystalysin cannot catabolize cysteine residues located within peptides. Cystalysin has Michaelis-Menten kinetics towards L-cysteine, and there was little or no inhibition by ammonia, H2S, pyruvate and acetate. Human erythrocytes incubated with H2S or with cystalysin and cysteine primarily accumulated sulfhemoglobin and methemoglobin, along with minor amounts of choleglobin and protein aggregates. Erythrocytes retained the ability to reduce methemoglobin in the presence of H2S. Cystalysin could not modify hemoglobin when beta-chloroalanine was the substrate, indicating an absolute requirement for H2S production. Cystalysin appears to be an unregulated L-cysteine catabolizing enzyme, with the resulting H2S production being essential to the atypical hemolytic activity.

Cystalysin, a 46-kDa L-cysteine desulfhydrase from Treponema denticola: biochemical and biophysical characterization.

A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67. Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics. Cystathionine and s-aminoethyl-L-cysteine were also substrates. Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin. The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase). Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T. denticola may also function in vivo as an important virulence molecule.

Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities.

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.

Core sugar residues of the N-linked oligosaccharides of Russell's viper venom factor X-activator maintain functionally active polypeptide structure.

We have previously showed that factor X activator of Russell's viper venom (RVV-X) contains six N-linked oligosaccharide chains: four in the heavy chain and one in each of the two light chains [Gowda, D.C., Jackson, C.M., Hensley, P., & Davidson, E.A. (1994) J. Biol. Chem. 269, 10644-10650]. In the present study, we have investigated the role of the carbohydrate moieties in the structure and functional activity of RVV-X. Sequential removal of sugar residues from the terminal ends by exoglycosidases, up to 50% of total carbohydrates, did not significantly alter the activity of RVV-X, demonstrating that the peripheral carbohydrate moieties are not involved in interactions with factor X. However, removal of whole oligosaccharide chains by N-glycanase caused an almost total loss of the ability of RVV-X to activate factor X to factor Xa. In parallel with these observations, circular dichroism spectroscopy showed that complete deglycosylation, but not the removal of peripheral sugars, caused a significant change in the secondary structure. Together, these data demonstrate that the oligosaccharide chains are necessary for the functional activity, and that the trimannosylchitobiose core residues are sufficient for the maintenance of the native polypeptide structure.

Noncooperativity of biotin binding to tetrameric streptavidin.

Streptavidin tetramers have been separated according to their biotin content by anion exchange chromatography. Biotin-free and biotin-saturated streptavidin were coincubated. Streptavidin at intermediate ligation levels, i.e., with one, two, or three molecules of bound biotin, accumulates over time. A steady state distribution of ligation levels is reached after 2 days. When biotin was allowed to redistribute starting from homogeneous populations containing two molecules of biotin per tetramer, a similar steady state distribution of ligation levels was observed, thereby demonstrating an equilibrium distribution. Quantification of this equilibrium indicates that biotin binds to streptavidin with no cooperativity.

Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese.

Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33,000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.

Purification of bovine liver rhodanese by low-pH column chromatography.

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.

The quaternary structure of streptavidin in urea.

We report on the interactions of urea and guanidinium salts with streptavidin. Gel filtration chromatography in 0, 4, 6, and 7 M urea indicates that the streptavidin tetramer remains intact in urea. Biotin alters the electrophoretic mobility of streptavidin whether or not 6 M urea is present. The intrinsic fluorescence of streptavidin is increased and blue-shifted in 6 M urea. The fluorescence changes indicate the absence of unfolding. A conformational response to urea is possible, but much of the fluorescence change is due to urea binding as a weak biotin analog (Ka approximately 1.3 M-1). The resistance to structural perturbation by urea reflects the structural stability of streptavidin's anti-parallel beta-barrel motif. Unfolding is sluggish in 6 M guanidinium hydrochloride (half-time, approximately 50 days). After guanidinium thiocyanate unfolding, streptavidin can be refolded, but the unfolding and refolding transitions are centered at different concentrations of perturbant. Slow unfolding, with a 15th power dependence on guanidinium thiocyanate concentration, may be partially responsible for the noncoincidence of the unfolding and refolding processes. Nonequilibrium behavior is also seen in 6 M urea, as native streptavidin does not unfold and guanidinium thiocyanate unfolded streptavidin does not refold. Refolding does occur at lower concentrations of urea. Guanidinium thiocyanate only slowly unfolds the biotin-streptavidin complex. In the presence of biotin, unfolded streptavidin does not refold in 6 M guanidinium thiocyanate or in 6 M urea.

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin.

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20-28% of the initial fluorescence with (KSV + V) less than or equal to 0.55 M-1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka = 5 M-1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weber et al., 1989).

NADPH-cytochrome P-450 reductase. Physical properties and redox behavior in the absence of the FAD moiety.

NADPH-cytochrome P-450 reductase contains one molecule each of FMN and FAD. The FAD moiety has been selectively removed, producing the FMN reductase. The FMN reductase is stable and enzymatic activity is reconstituted with either FAD or FMN. FMN remains tightly bound, but can both dissociate from the FMN site and bind to the vacant FAD site. The amount of FMN bound in the FAD site is minimal under specific experimental conditions. There are at least two conformational subpopulations of the FMN reductase; NADP dissociates readily from one but extremely slowly from the other. Rapid dissociation of NADP is regained upon reconstitution with FAD. The one-electron redox state of the FMN reductase is thermodynamically stabilized, though to a lesser degree than in the holoreductase. When two-electron reduced FMN reductase is exposed to oxygen, a stable species with an absorbance peak at 580 nm forms rapidly and quantitatively. This species has been identified by electron paramagnetic resonance spectroscopy as the neutral radical of FMN and is indistinguishable from the air-stable radical of the holoreductase. The redox behavior of the FMN reductase is in agreement with properties proposed previously for the FMN site.

Shielding of tryptophan residues of avidin by the binding of biotin.

The binding of biotin to tetrameric avidin changes the environment of tryptophan residues. Binding reduces the total tryptophan fluorescence by 34%, shifts the emission peak from 337 to 324 nm, and reduces the fluorescence bandwidth from 61 to 46 nm. These changes are consistent with the movement of tryptophans to a nonpolar, internal environment. In the absence of biotin, iodide readily quenches the fluorescence of 20-29% of the initial fluorescence, which likely corresponds to one tryptophan located in a positively charged environment. Iodide may have weak access to additional fluorescence, corresponding to perhaps one additional tryptophan. Acrylamide, in the absence of biotin, has good access to three-fourths or more of the fluorescence, but the remainder, due to one or two tryptophans, is well shielded. The binding of biotin completely prevents iodide quenching and decreases acrylamide access dramatically. The data indicate that biotin binding shifts two or three tryptophans to an internal, hydrophobic, shielded environment.

Giant polypeptides of skeletal muscle titin: sedimentation equilibrium in guanidine hydrochloride.

Titin has been purified from rabbit skeletal myofibrils in guanidine hydrochloride by gel filtration on Sephacryl S-1000. The sedimentation coefficients of denatured titin in 6 M guanidine hydrochloride are highly concentration dependent and follow the equation: 1/S = 0.241 + 0.302 A280. Equilibrium sedimentation studies indicate that the average molecular weight of titin polypeptides is 2.4 to 2.6 X 10(6).

Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase.

NADPH-cytochrome P-450 reductase releases FAD upon dilution into slightly acidic potassium bromide. Chromatography on high performance hydroxylapatite resolved the FAD-dependent reductase from holoreductase. The FAD dependence was matched by a low FAD content, with the ratio of FAD to FMN as low as 0.015. The aporeductase had negligible activity toward cytochrome c, ferricyanide, menadione, dichlorophenolindophenol, nitro blue tetrazolium, and an analogue of NADP, acetylpyridine adenine dinucleotide phosphate. A 4-min incubation in FAD reconstituted from one-half to all of the enzyme activity, as compared to the untreated reductase, depending upon the substrate. After a 2-h reconstitution, the reductase eluted from hydroxylapatite at the same location in the elution profile as did the untreated holoreductase. The reconstituted reductase had little flavin dependence, was nearly equimolar in FMN and FAD, and had close to the specific activity, per mol of flavin, of untreated reductase. The dependence upon FAD implies that FMN is not a competent electron acceptor from NADPH. Thus, the FAD site must be the only point of electron uptake from NADPH.