The iron and subunit binding sites of hemerythrin. The role of histidine, tyrosine and tryptophan.

Of the three tyrosine residues available for nitration by tetranitromethane in hemerythrin, nitration of tyrosine residue 70 has no effect on dissociation of octomers to monomers, but nitration of tyrosines 18 and/or 67 results in dissociation to monomers. The latter data suggests these residues are important for subunit association. The reactive sulfhydryl, the modification of which produces dissociation, was protected as a mixed disulfide during the nitration but was regenerated for analysis of the state of association. Residue 70 can be selectively modified because of its exposed position and perhaps because of its slightly lower pk of 6.9, compared to 7.3 as an average of all nitrotyrosines in a completely nitrated hemerythrin. Solvent perturbation studies in 20% Me2SO indicate that 3 tyrosines, in agreement with the nitration results, and 2 tryptophan residues are exposed; however, oxidation at a 2-fold molar excess of N-bromosuccinimide oxidizes three tryptophan whereas a 3.5-fold excess oxidizes all four, but results in a rapid active site destruction. Photo-oxidation with methylene blue results in oxidation of only two tryptophan residues. These data have been interpreted to indicate that two tryptophans are free and two are involved in subunit association. Photo-oxidation with methylene blue results in the destruction of three histidines but no decrease in active site absorption. Histidine modification with diethyloxydiformate shows that three histidines react with no change in active site absorption. These results indicate that four histidines are unreactive toward these modifying agents and are therefore either buried or are ligands to the iron.     

The involvement of one of the three histidine residues of cow kappa-casein in the chymosin-initiated milk clotting process.

Cow kappa-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxy-formylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of kappa-casein to chymosin hydrolysis. Treatment of kappa-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow kappa-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.     

Tryptophanyl and carboxylic acid residues in the a centre of glucoamylase I from Aspergillus niger

The pH-dependence of the photo-oxidation of L-tryptophan, in the presence of Rose Bengal and Methylene Blue, has been investigated. True, initial rate constants were determined in order to circumvent errors due to secondary processes. Photo-oxidation of glycoamylase I from A. niger in the presence of Methylene Blue or Rose Bengal resulted in a pH-dependent loss of enzymic activity, which was analogous to the destruction of free L-tryptophan during photo-oxidation. The loss of enzymic activity was closely associated with the destruction of tryptophan residues in the enzyme. Significant protection of both enzymic activity and tryptophanyl residues in the enzyme molecule was achieved by performing the photo-oxidation in the presence of maltose, which is a substrate for the enzyme. The tryptophanyl residues of glucoamylase I, which had been inactivated by reaction of its carboxylic acid residues with glycine methyl ester in the presence of a water-soluble carbodi-imide, were also substantially protected by maltose. It is concluded that the active centre of glucoamylase I is a cleft lined with tryptophanyl residues that participate in the binding of the substrate. One or more carboxylic acid residues are involved in bond cleavage.     

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.     

An exposed tyrosine residue of RNase T1 and its involvement in the interaction with guanylic acid

A photo-CIDNP spectrum of RNase T₁ showed that 4 out of the total 9 tyrosines are accessible to the photosensitive dye, while none of the histidine and tryptophan residues are accessible. By comparison with the results of nitration of tyrosine side chains followed by peptide analysis, it can be concluded that Tyr 45 is mostly exposed on the surface of RNase T₁. On addition of 2'-GMP, the signal of Tyr 45 shifts upfield and is remarkably broadened, which suggests that the phenyl ring of Tyr 45 stacks on the guanine ring of 2'-GMP. Similar phenomena were observed on addition of 3'-GMP and 3'-dGMP.     

Gel filtration studies on oxyhemerythrin. II. Effect of temperature and ionic strength on the association-dissociation equilibria.

The effects of temperature and ionic strength on the association of oxyhemerythrin have been studied. deltaH degrees and deltaS degrees for association at pH 7.0 are -2.6 kcal and +16.5 eu per mol of monomer. These values suggest that solvent adjacent to the surface of the protein undergoes rearrangement on association. Increasing ionic strength is observed to promote dissociation while decreasing the rate of attainment of equilibrium between monomers and octamers. Qualitatively similar results are observed on lowering the pH from 7.0 to 4.8, thereby linking the effects of increasing ionic strength to those of protonation of specific amino acid residues at the subunit contacts of hemerythrin. The apparent enthalpy of ionization of the amino acid residue controlling dissociation at acidic pH was found to be -1.9 to +2.1 kcal/mol. These values are consistent with a carboxyl group.     

Complete chemical modification of the car☐yl groups in hemerythrin: Effect on the active center and subunit interactions

Chemical modification of all 18 car?yl groups in Golfingia gouldii hemerythrin was achieved using glycine methyl ester and 1-ethyl-3-dimethyl-aminopropylcarbodiimide. The absorption spectrum of the modified protein in the visible and near ultraviolet region was the same as that of unmodified hemerythrin, indicating that car?yl groups are not linked to iron in the active center. Modification of all 18 or as few as 8 car?yl groups resulted in dissociation of the octameric hemerythrin into monomers. Analysis of mixtures of octamer and monomer produced by partial modification suggest the involvement of one specific car?yl group in the observed dissociation reaction.     

Human placental alkaline phosphatase: Effects on conformation by ligands which alter catalytic activity

The conformation of human placental alkaline phosphatase (EC 3.1.3.1) has been studied using the spectroscopic structural probes of pH difference spectroscopy, solvent perturbation difference spectroscopy, and circular dichroism. Of the 37 ± 1 tyrosine residues in placental alkaline phosphatase (PAP), 5 ± 1 residues are observed by pH difference spectroscopy to be “free” and presumed to be located on the surface of the enzyme molecule. The ionization of these 5 “free” tyrosyl groups is not time dependent and is reversible with a pK_app of 10.29. The remaining 32 ± 1 tyrosines are considered “buried” and ionization is observed to be both time dependent and irreversible. Treatment of the enzyme with 4 m guanidine-hydrochloride normalizes all 37 ± 1 tyrosine residues (pK_app = 10.08). The difference pH titration studies thus provide spectrophotometric evidence for a change in molecular conformation of PAP in the pH region of 10.5. Using solvent perturbation difference spectroscopy and circular dichroism, the local environments of tyrosine and tryptophan residues were elucidated for the native enzyme and the enzyme in the presence of ligands that influence catalytic function: inorganic phosphate (competitive inhibitor), l-phenylalanine (uncompetitive inhibitor), d-phenylalanine (noninhibitor). and Mg²⁺ ion (activator). The spectral observations from these studies led to the following interpretations: (i) the binding of inorganic phosphate, a competitive inhibitor, induces a conformational change in the enzyme that may alter the active site and thereby decrease enzyme catalytic function; (ii) perturbation with l-phenylalanine gives spectral results indicating a conformational change consistent with the postulate that this uncompetitive inhibitor prevents the dissociation of the phosphoryl enzyme intermediate; and (iii) Mg²⁺ ion causes a slight separation of the enzyme subunits, which could increase accessibility to the active site and, thus, enzyme activity.     

Reaction of bovine and equine growth hormones with tetranitromethane.

Bovine and equine growth hormones were chemically modified with tetranitromethane, at pH 7.4 during 5 h and at pH 8.0 in the presence of 8 M urea during 1 h. a) Both hormones have very similar but not identical reactivities. b) The nitration of the reactive tyrosines and tryptophan residues at pH 7.4 produces no detectable changes in their immunological or somatotrophic activities. C) The nitration of all tyrosine residues in both hormones gives rise to a complete loss of somatographic activity with no alteration of the immunological activity.     

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

The active site of the catalytic domain of stromelysin-1 (matrix metalloproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the environmentally sensitive fluorescent reporter molecule bisANS. Wavelength-dependent acrylamide quenching identified three distinct emitting tryptophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Significant changes in the tryptophan fluorescence polarization occur upon binding by any of the three hydroxamate inhibitors Batimastat, CAS108383-58-0, and Celltech CT1418, all of which bind in the P2′-P3′ region of the active site. In contrast, the inhibitor CGS27023A, which is t hought to bind in the P1-P1′ region, does not induce any change in tryptophan fluorescence polarization. The use of the fluorescent probe bisANS revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysin with a dissociation constant ofK _i=22 μM. In addition to this binding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40±0.17 μM. The auxiliary site is open, hydrophobic, and near the fluorescing tryptophans. The binding of bisANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.     

Acetylcholinesterase: inhibition by tetranitromethane and arsenite. Binding of arsenite by tyrosine residues

Tetranitromethane inhibits acetylcholinesterase with respect to the hydrolysis of both acetylthiocholine and indophenyl acetate. The loss of activity with indophenyl acetate, a poor substrate, is preceded by an increase in enzyme activity. Only 12 of the 21 tyrosine residues/monomer of enzyme are susceptible to nitration. Loss of activity with respect to indophenyl acetate occurs well after no further nitration of tyrosines occurs and must be due to the modification of other residues. Incubation of the enzyme with arsenite before nitration results in the nitration of only 10 tyrosines. This experiment reveals that the structural basis for the binding of arsenite is the formation of a diester with two tyrosine residues.     

Functionality of nitrated acetylcholine receptor: the two-step formation of nitrotyrosines reveals their differential role in effectors binding

The presence of nitrotyrosines is associated with several neurodegenerative pathologies. We evaluated the functionality of the nicotinic acetylcholine receptor possessing nitrotyrosines. The spectrum of the nitrated receptor displays an absorption band characteristic of ortho-nitrophenol. The presence of carbamylcholine in the agonist site prevented the effect of nitration by tetranitromethane in some conditions. The nitration occurred with two discrete steps and pointed out the differential involvement of tyrosines in the binding of acetylcholine and neurotoxin. We concluded that at least two residues involved in agonist binding can be nitrated, which bring similar contributions to the binding energy of the neurotransmitter.     

Peroxynitrite and fibrinolytic system: The effect of peroxynitrite on plasmin activity

We have shown that peroxynitrite (ONOO-) inhibits streptokinase-induced conversion of plasminogen to plasmin in a concentration-dependent manner and reduces both amidolytic (IC5o approximately 280 microM at 10 microM concentration of enzyme) and proteolytic activity of plasmin. Spectrophotometric and immunoblot analysis of peroxynitrite-treated plasminogen demonstrates a concentration-dependent increase in its nitrotyrosine residues that correlates with a decreased generation of active plasmin. Peroxynitrite (1 mM) causes the nitration of 2.9 tyrosines per plasminogen molecule. Glutathione, like deferoxamine, partially protects plasminogen from peroxynitrite-induced inactivation and reduces the extent of tyrosine nitration. These data suggest that nitration of plasminogen tyrosine residues by peroxynitrite might play an important role in the inhibition of plasmin catalytic activity.     

Two forms of trehalase in rabbit enterocyte: Purification and chemical modification

Trehalase found to be associated with the brush border membrane vesicles and the Ca₂₊ aggregated basolateral membrane vesicles were purified to homogeneity. They were found to differ in their molecular weight, subunit structure, heal stability, N-terminal residues, amino acid composition and also the active site residues. Chemical modification showed the presence of a histidine and tyrosine at the active site of brush border membrane vesicle trehalase and two histidines at the active site of basolateral membrane vesicle.     

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

The active site of the catalytic domain of stromelysin-1 (matrix metalloproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the environmentally sensitive fluorescent reporter molecule bisANS. Wavelength-dependent acrylamide quenching identified three distinct emitting tryptophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Significant changes in the tryptophan fluorescence polarization occur upon binding by any of the three hydroxamate inhibitors Batimastat, CAS108383-58-0, and Celltech CT1418, all of which bind in the P2′-P3′ region of the active site. In contrast, the inhibitor CGS27023A, which is t hought to bind in the P1-P1′ region, does not induce any change in tryptophan fluorescence polarization. The use of the fluorescent probe bisANS revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysin with a dissociation constant ofK _i=22 μM. In addition to this binding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40±0.17 μM. The auxiliary site is open, hydrophobic, and near the fluorescing tryptophans. The binding of bisANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.     

Urea-induced inactivation, dissociation, and unfolding of the allosteric phosphofructokinase from Escherichia coli

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.     

Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.     

Evidence for essential histidine and dicarboxylic amino-acid residues in the active site of UDP-glucose : solasodine glucosyltransferase from eggplant leaves

Effects of several chemical probes selectively modifying various amino-acid residues on the activity of UDP-glucose : solasodine glucosyltransferase from eggplant leaves was studied. It was shown that diethylpyrocarbonate (DEPC), a specific modifier of histidine residues, was strongly inhibitory. However, in the presence of excessive amounts of the enzyme substrates, i.e. either UDP-glucose or solasodine, the inhibitory effect of DEPC was much weaker indicating that histidine (or histidines) are present in the active site of the enzyme. Our results suggest also that unmodified residues of glutamic (or aspartic) acid, lysine, cysteine, tyrosine and tryptophan are necessary for full activity of the enzyme. Reagents modifying serine and arginine residues have no effect on the enzyme activity.     

An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.