Role of arginine and histidine residues in the biological activity of botulinic neurotoxin A

Treatment of botulinic neurotoxin A with cyclohexanedione demonstrated that modification of 5 to 10 arginine residues does not change the neurotoxin toxicity, while after modification of 15-20 arginine residues the toxicity is decreased by 40-50% of the original value. Butanedione exerts a stronger detoxicating effect on neurotoxin than cyclohexanedione. The molecular conformation of the modified toxin derivatives and their precipitability upon interaction with antisera against toxin and toxin fragments does not change thereby. The non-toxic derivatives of toxin containing 40 modified arginine residues possess a partial serological affinity for the original toxin in a reaction with antiserum against toxin but do not interact with the antifragment sera. The molecular conformation of these preparations is changed considerably. It is assumed that one or two arginine residues are located near the toxic site of the neurotoxin molecule and are also components of its antigenic determinants. Modification of histidine residues in the neurotoxin molecule by diethylpyrocarbonate is accompanied by a decrease of its toxicity. An additional 10% toxicity is revealed upon modification of 11-13 histidine residues. The molecular conformation of the modified derivatives of neurotoxin and their precipitability do not change thereby. It is probable that 1 or 2 histidine residues are located at or near the toxic site. The data obtained suggest that histidine residues are not localized in antigenic determinants of the neurotoxin molecule.     

Effects of histidine modification on the biological and immunological activities of equine chorionic gonadotropin

The histidine residues of equine chorionic gonadotropin have been modified and the resultant effects on the biological and immunological activities of the hormone examined. The modification of histidine is carried out by rose bengal-sensitized photooxidation. The kinetics of histidine destruction is biphasic, a rapid loss followed by a slower decrease. An average loss of 15–20% of the total histidines in the molecule resulted in 70–80% disappearance of the lutropin activity with no significant loss in the immunological activity. The loss of lutropin activity paralleled the decline of follitropin activity. Further destruction of histidine of up to 70–90% of the total resulted in over a 99% loss of biological activity with no significant effect on the immunological activity. Modified equine chorionic gonadotropin was unable to compete with the native molecule in the rat Leydig cell assay. These observations suggest that the immunorecognition site in equine chorionic gonadotropin is different from the region responsible for the biological activity and does not involve a histidine residue. Furthermore, it also appears that the histidine residues are important for the manifestation of both lutropin and follitropin activities.     

Photochemical oxidation of snake gourd proteinase A2, a serine proteinase

Snake gourd proteinase A₂ was rapidly inactivated by methylene blue catalysed photooxidation at pH 7.8 and 25°. The rate of inactivation was pH-dependent and became slower at lower pH values, suggesting the involvement of some histidine residues in the inactivation. Changes in amino acid composition occurred only with histidine residues. One mole or more of histidine residues in the molecule are of essential importance in the catalytic function of snake gourd proteinase A₂.     

Ethoxyformylation of ribonuclease U2 form Ustilago sphaerogena.

RNase U2 was inactivated by incubation with ethoxyformic anhydride at pH 6.0 and pH 4.5. The absorbance of the RNase U2 increased at around 250 nm and decreased at around 280 nm. The inactivation occurred in parallel with the amount of modified histidine and plots of the relationship between the remaining activity and the modified histidine suggested that the modification of one of the two histidine residues totally inactivated the enzyme. The inactivated enzyme RNase U2 was reactivated by a low concentration of hydroxyamine, with removal of the ethoxyformyl group from the modified histidine residue. At pH 4.5, 2'-adenylate and 2'-guanylate protected RNase U2 from inactivation by ethoxyformic anhydride. The difference CD spectra showed that the ability of RNase U2 to form a complex with 2'-adenylate was lost on ethoxyformylation.     

Light-induced protein conformational changes in the photolysis of octopus rhodopsin.

Light-induced protein conformational changes in the photolysis of octopus rhodopsin were measured with a highly sensitive time-resolved transient UV absorption spectrophotometer with nanosecond time resolution. A negative band around 280 nm in the lumirhodopsin minus rhodopsin spectra suggests that alteration of the environment of some of the tryptophan residues has taken place before the formation of lumirhodopsin. A small recovery of the absorbance at 280 nm was observed in the transformation of lumirhodopsin to mesorhodopsin. Kinetic parameters suggest that major conformational changes have taken place in the transformation of mesorhodopsin to acid metarhodopsin. In this transformation, drastic changes of amplitude and a shift of a difference absorption band around 280 nm take place, which suggest that some of the tryptophan residues of rhodopsin become exposed to a hydrophilic environment.     

Essential histidine residues in coenzyme B12-dependent diol dehydrase: dye-sensitized photooxidation and ethoxycarbonylation

The apoenzyme of diol dehydrase was inactivated by photoirradiation in the presence of rose bengal or methylene blue, following pseudo-first-order kinetics. The inactivation rates were markedly reduced under a helium atmosphere, suggesting that the inactivation is due to photooxidation of the enzyme under air. The half-maximal rate of methylene blue-sensitized photoinactivation was observed at pH around 7.5. Amino acid analyses indicated that one to two histidine residues decreased upon the dye-sensitized photoinactivation, whereas the numbers of tyrosine, methionine, and lysine did not change. Ethoxyformic anhydride, another histidine-modifying reagent, also inactivated diol dehydrase, with pseudo-first-order kinetics and a half-maximal rate at pH 7.7. It was shown spectrophotometrically that three histidine residues per enzyme molecule were modified by this reagent with loss of enzyme activity. Two tyrosine residues per enzyme molecule were also modified rapidly, irrespective of the activity. The photooxidation or ethoxycarbonylation of the enzyme did not result in dissociation of the enzyme into subunits, but deprived the enzyme of ability to bind cyanocobalamin. The percentage loss of cobalamin-binding ability agreed well with the extent of inactivation. The enzyme-bound hydroxocobalamin showed only partial protecting effect against photoinactivation and resulting loss of the cobalamin-binding ability. These results provide evidence that diol dehydrase possesses essential histidine residues which are required for the coenzyme binding.     

Photooxidation of human serum albumin and its complex with bilirubin.

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen. Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected. It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.     

Role of histidine residues in ovine lutropin: effects on steroidogenic activity.

The modification of histidine residues of ovine pituitary lutropin by rose bengal sensitized photooxidation has been investigated. The destruction of an average of one histidine out of six lead to 90% loss of biological activity as examined by the $\text{in}\text{vitro}$ steroidogenic response in the rat Leydig cell essay. Further modification of an average 2 – 3 histidine residues reduced the biological activity to less than 1% of the native lutropin. The modified lutropin was incapable of inhibiting the native lutropin induced steroidogenesis. Gel filtration experiments and polyacrylamide disc gel electrophoresis patterns indicated that no dissociation of the molecule into subunits occurred. This is the first report on the essentiality of the histidine residue for the activity of lutropin.     

Histidine at the active site of Neurospora tyrosinase

The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation.     

Effect of diethylpyrocarbonate on the biological activities of botulinum neurotoxin types A and E

The single chain (unnicked) type-E and the dichain (nicked) type-A botulinum neurotoxins were modified with diethylpyrocarbonate (ethoxyformic anhydride), a reagent highly specific for histidine residues. The type-E neurotoxin could be completely detoxified without causing detectable damage to its serological reactivity. Under identical modification reaction conditions, the type A was incompletely detoxified with some alteration in its serological reactivity. Modification of histidine residues was evident from the increase in absorbance at 240 nm, and reactivation of the detoxified proteins by reversing the modification with hydroxylamine. The completely detoxified type-E neurotoxin, used as toxoid, elicited antibodies in rabbits. The antiserum precipitated and neutralized the neurotoxin. This toxoid is homogeneous as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the traditional toxoid produced with formaldehyde is heterogeneous.     

The specific modification of histidyl residues of inorganic pyrophosphatase from Bacillus stearothermophilus by photooxidation

Photooxidation of inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] from Bacillus stearothermophilus in the presence of rose bengal resulted in rapid loss of enzymatic activity. The pH profile of the inactivation rate by the photooxidation showed an inflection point around pH 6.8, suggesting the involvement of histidyl residues in the inactivation. Amino acid analysis revealed that the loss of enzymatic activity was accompanied by the destruction of 3 histidyl residues per molecule. The presence of Mg2+ alone afforded partial protection against the inactivation, whereas inorganic pyrophosphate, the substrate, showed almost no protective effect against inactivation. The photooxidation of inorganic pyrophosphatase altered the circular dichroism spectrum and the difference UV spectrum induced by Mg2+ in the near ultraviolet region. These results suggested that histidyl residues appear to be located at the binding site of Mg2+ and may contribute to the conformational change induced by Mg2+.     

Evidence for essential histidine residues in bovine-liver mitochondrial monoamine oxidase.

Ethoxyformic acid anhydride, amino-1H-tetrazole, and photooxidation in the presence of rose bengal, which are reagents known to react with histidine residues of proteins, were shown to inactivate monoamine oxidase. Ethoxyformic acid anhydride reacted with about 6 histidine residues per 100 000 g of protein under the experimental conditions adopted and completely inactivated the enzyme. However, NH2OH reactivated the ethaxyformic acid derivative t only. Since NH2OH specifically deacylates N-ethoxyformylimidazole, it was shown that at least some of the histidine residues are essential for activity. In addition, photooxidation experiments in the presence of 0.01% rose bengal confirmed that only histidine residues of bovine hepatic monoamine oxidase are destroyed under the designated experimental conditions. About 9 histidine residues per 100 000 g of protein were destroyed during the photooxidation experiments. In the presence of substrate, kynuramine or benzylamine, only 7 histidine residues were destroyed, which indicates that 2 histidine residues per 100 000 g of protein are essential for activity.     

Serratia marcescens endonuclease. Properties of the enzyme

Some physico-chemical properties of endonuclease (EC 3.1.4.9) from Serratia marcescens were studied and the amino acid composition of the enzyme was determined. The protein molecule was shown to contain one SH-group and one S-S-bond, which renders it different from the well studied nuclease (EC 3.1.4.7) from Staph. pyogenes. The conditions for reconstitution of the S-S-bond by dithioerythritol for quantitative estimation of cysteine residues of the endonuclease molecule were selected. The N-terminal amino acid was found to be threonine. The UV spectra for the enzyme are typical for proteins; A 0,1% 1cm,280nm is 1.46, epsilon 25 degrees 280nm,pH7,4 is 47292 M-1 cm-1. The sedimentation coefficient in phosphate buffer sW, 20 degrees is 3.4 S, pI is 6.5 and 7.5.     

Reversible inhibition of the mitochondrial ubiquinol-cytochrome c oxidoreductase complex (complex III) by ethoxyformic anhydride

The mitochondrial ubiquinol-cytochrome c oxidoreductase (complex III) is inhibited by ethoxyformic anhydride (EFA). The inhibition is readily reversed by hydroxylamine, suggesting the involvement of essential histidyl or possibly tyrosyl residues. The spectrum of ethoxyformylated complex III in the UV region showed a peak at 238 nm, indicative of N-(ethoxyformyl)histidine. Addition of hydroxylamine caused a large decrease of the 238-nm peak, which amounted to 16 mol of (ethoxyformyl)histidine/mol of cytochrome c1. Hydroxylamine addition to ethoxyformylated complex III also caused a small change at about 280 nm, which could be due to reversal of 1.6 O-ethoxyformylated tyrosyl residues/mol of cytochrome c1. Among many inhibitors of the cytochrome bc1 region of the respiratory chain, EFA is the only reagent known to cause reversible inhibition by covalent modification of amino acid residues. The inhibition site of EFA was determined to be between cytochromes b-562 and c1. However, unlike antimycin, which also inhibits in the same region, EFA did not promote the reduction of cytochrome b-566 in particles treated with substrates. In addition, it was found that EFA inhibits proton translocation in the cytochrome bc1 region and is a more effective electron transport inhibitor when added to reduced particles as compared to oxidized particles. These results together with the strong possibility that the EFA target is a histidyl or possibly a tyrosyl residue have been discussed in relation to the mechanism of proton translocation by complex III.     

Step-wise thermal denaturation of cobrotoxin, a snake venom neurotoxin fromNaja naja atra: A proton nuclear magnetic resonance study

Temperature dependence of proton nuclear magnetic resonance spectra has been followed for cobrotoxin, a postsynaptic neurotoxin fromNaja naja atra venom. Several aromatic amino-acid residues, including the functionally essential Trp-29 located at the tip of the central loop of the molecule, have been found to undergo a thermal structural transition above the global thermal denaturation temperature. It is suggested that a local structure around these residues behaves somehow independently of the rest of the molecule, and that such structural organization may be favorable for a conformational change of a neurotoxin molecule on binding to acetylcholine receptor.     

Step-wise thermal denaturation of cobrotoxin,a snake venom neurotoxin fromNaja naja atra: A proton nuclear magnetic resonance study

Temperature dependence of proton nuclear magnetic resonance spectra has been followed for cobrotoxin, a postsynaptic neurotoxin fromNaja naja atra venom. Several aromatic amino-acid residues, including the functionally essential Trp-29 located at the tip of the central loop of the molecule, have been found to undergo a thermal structural transition above the global thermal denaturation temperature. It is suggested that a local structure around these residues behaves somehow independently of the rest of the molecule, and that such structural organization may be favorable for a conformational change of a neurotoxin molecule on binding to acetylcholine receptor.     

Interaction of rabbit hemopexin with rose bengal and photooxidation of the rose bengal-hemopexin complex.

Rabbit hemopexin associates with rose bengal producing a hypochromic shift in the absorption spectrum of the dye; the extinction coefficient of the dye bound to heme-saturated hemopexin is approximately 20% lower than that of the dye bound to the apoprotein. The interaction of apo- and heme-saturated hemopexin with rose bengal was studied in detail by difference spectroscopy. Apo-hemopexin has one tight binding site for the dye with a dissociation constant in the micromolar range and a set of several weaker binding sites. In contrast, heme-saturated hemopexin has a very low affinity for the dye. Evidence that histidine residues of hemopexin participate in the binding of heme was obtained by photooxidation of hemopexin sensitized by rose bengal. Progressive modification of the 16 histidine residues of hemopexin is effected by illumination of the dye-hemopexin complexes. The midpoint of this pH-dependent reaction is at pH 6.8 +/- 0.1. In 15 min of irradiation, apo-hemopexin loses 50% of its ability to form a low spin hemichrome complex with deuteroheme while only 10% of the ligand coordination to heme iron of the deuteroheme-hemopexin is lost. At that time, approximately 2 more histidine residues are modified in apo-hemopexin than in deuteroheme-hemopexin, and no change is found in other potentially photolabile amino acid residues. The characteristic circular dichroism positive extremum at 231 nm of hemopexin also was decreased by photooxidation, and the loss was slower in the deuteroheme-hemopexin complex than in the apoprotein. When deuteroporphyrin IX was used as the photosensitizing agent, similar results were obtained.     

A kinetic study of the photochemical inactivation of adenylate kinases of Mycobacterium marinum and bovine heart mitochondria

Using incident light energy of about 76 mW.cm-2 in a dye-sensitized photooxidation reaction, we have investigated the possible involvement of one or both of the histidine residues in the catalytic activity of adenylate kinase (ATP:AMP phosphotransferase) of Mycobacterium marinum. We have done this by investigating the kinetics of photochemical inactivation of the enzyme. At pH 7.4, the kinetics of photoinactivation are biphasic with two different pseudo-first-order rate constants. Adenosine 5'-pentaphospho 5'-adenosine (Ap5A), ATP and, to some extent, AMP, all gave protection to the enzyme from inactivation. Amino-acid analysis of the photoinactivated enzyme indicated the loss of the two histidine residues. This, and the fact that photoinactivation occurred faster at alkaline compared to acidic pH, indicated the involvement of the histidine residues in the catalytic activity. A mathematical model is developed which assumes that both histidine residues are required for maximal catalytic activity: one is located peripherally, is exposed, and therefore is readily photooxidized (pseudo-first-order rate constant, k1 = 1.3.10(-2)s-1), while the other is located at the active site, involved in substrate-binding and is shielded (pseudo-first-order rate constant, k2 = 2.9.10(-4)s-1). However, this shielded histidine could be exposed and made more accessible to photooxidation either by raising the pH above 10, or alternatively, by the addition of 8 M acetamide (or 6 M guanidine). Under these conditions, which apparently cause unfolding of the protein molecule, the kinetics of photoinactivation change from biphasic to monophasic, suggesting that both histidine residues are equally exposed and are photooxidized at the same rate. Unlike the enzyme from M. marinum, adenylate kinase from bovine heart mitochondria shows monophasic kinetics of photoinactivation at pH 7.4, suggesting that only one of the six histidine residues is essential for catalytic activity, or if more than one, then they all must be equally exposed. Further, ATP, AMP or Ap5A did not provide protection against photoinactivation, suggesting that the histidine residue(s) involved in the catalytic activity must remain exposed after the substrates bind at the active site of the mitochondrial enzyme.     

Photochemical Yields in Ribonuclease and Oxidized Glutathione Irradiated at Different Wavelengths in the Ultraviolet

The quantum yields for the disruption of various amino acids in glutathione and ribonuclease by 229, 254, 265, and 280 nm UV photons have been determined. The results of the measurements on the destruction of tyrosine and histidine and the loss of enzymic function in RNAse and the disruption of cystine in both compounds lead to the following conclusions: (a) The photodestruction of some and perhaps many constituent amino acid residues does not cause RNAse inactivation. (b) Contrary to the basic premise of proposals made by other authors, the photochemical yields of constituent residues in a protein are not the same as that for the same amino acids in solution alone-the difference is a function of the exciting wavelength. Further, the extent of histidine destruction varies by a large factor among three proteins. (c) Consistent with previous predictions, the present results show that photons absorbed in the aromatic residues of RNAse cause the disruption of cystines elsewhere in the enzyme. (d) Although cystine disruption appears to be the most prevalent mode of RNAse inactivation by photons of the four wavelengths studied, some of the minor mechanisms leading to loss of enzymic function may vary with the UV energy.     

Studies on the toxin of Aspergillus fumigatus. XVIII. Photooxidation of asp-hemolysin in the presence of various dyes and its relation to the site of hemolytic activity

The site of hemolytic activity of a toxin isolated from Aspergillus fumigatus designated Asp-hemolysin was determined by photooxidation techniques. The hemolytic activity of this toxin was strongly inhibited by photooxidation with methylene blue, rose bengal, riboflavin, or eosin G as a sensitizer, whereas crystal violet, hematoxylin, naphthol yellow S, bromothymol blue, methyl orange, and cresol red had no effect. pH dependence of the inactivation with methylene blue was observed in the narrow range of pH values from 7.0 to 8.0, like that of the inactivation with rose bengal or riboflavin. The histidine, cysteine, methionine, tryptophan, and tyrosine content of methylene blue-photooxidized Asp-hemolysin was significantly decreased, while other amino acids were not affected. The hemolytic activity of the toxin was lost more slowly than the histidine residue, being maintained at about 50% even at the time when the histidine residue was completely lost after 30 min. Photooxidation of Asp-hemolysin in the presence of rose bengal also caused a decrease in histidine, methionine, and threonine content. These findings suggest that residues of cysteine, methionine, threonine, tryptophan, and/or tyrosine but not histidine may play an important role through stereostructure in the manifestation of the hemolytic activity of Asp-hemolysin.