Consequences of the modification of tryptophan-215 on the function of bovine alpha-chymotrypsin

At pH values between 4.5 and 7.0, 2-hydroxy-5-nitrobenzyl bromide reacts selectively with tryptophan-215 in bovine α-chymotrypsin as demonstrated by the isolation of peptides containing modified amino acid residues. The degree of substitution at lower pH values indicates conformational changes in the enzyme observed previously by physico-chemical methods. The substitution of the native enzyme results in the loss of esterase activity. Nevertheless 2-hydroxy-5-nitro-benzyl chymotrypsin is still able to react with diisopropylphosphofluoridate.The catalytically inactive derivatives of α-chymotrypsin, DIP, TPCK and anhydro-chymotrypsin, as well as the complex of α-chymotrypsin with basic pancreatic trypsin inhibitor, are not modified by 2-hydroxy-5-nitrobenzyl bromide under the same conditions as those used for the native enzyme.HNB-chymotrypsin and anhydro-chymotrypsin, both catalytically inactive, form stoichiometric complexes with the basic pancreatic trypsin inhibitor whereas both PMS and DIP α-chymotrypsin did not have this complexing property. From the results of this and a preceding study (Ako et al., 1972) it is concluded that the intactness of the catalytic function of ehymotrypsin is not obligatory for the binding of basic pancreatic inhibitor.     

Structure and action of heteronemertine polypeptide toxins: Inactivation of Cerebratulus lacteus toxin B-IV concomitant with tryptophan alkylation

Reaction of Cerebratulus lacteus toxin B-IV with 2-hydroxy-5-nitrobenzyl bromide at pH 4.5 results in modification of toxin tryptophan residues and loss of biological activity. With relatively small reagent excesses, one tryptophan per molecule is modified without major effect on toxicity. Further reaction results in modification of a second residue of tryptophan and loss of at least 95% of the toxic activity. Modification of one or both tryptophan residues is without significant effect on the secondary structure of the protein. The specificity of each phase of the reaction has been assessed by fingerprint analysis of peptides derived from toxin modified to differing extents with 2-hydroxy-5-nitrobenzyl bromide. It is thus possible to show that tryptophan-5 reacts first and tryptophan-30 only under more rigorous conditions. It thus appears that tryptophan-30 is essential for full neurotoxic activity.     

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1

Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122-204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.     

The modification of tryptophan in bovine thrombin.

2-Hydroxy-5-nitrobenzyl bromide, at a 100-fold molar excess, was observed to react withthrombin at pH 4.0 to give a modified enzyme which possessed 20% of the fibrinogen clotting activity and 80% of the esterase activity compared to a control preparation. Spectrophotometric analysis of the modified protein indicated that this effect on catalytic activity was associated with the incorporation of 1 mol of reagent per mol of thrombin. Amino acid analysis showed no loss of amino acids other than tryptophan. The reaction of N-bromosuccinimide with thrombin at 2-fold molar excess resulted in the modification of one tryptophan per mol of enzyme with the loss of 80% of the fibrinogen clotting activity with, as above, a considerably smaller loss of esterase activity. Oxidation of thrombin with N-bromosuccinimide decreased the extent of subsequent tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Thrombin modified with 2-hydroxy-5-nitrobenzyl bromide showed a 3-4 fold increase in Km and a decrease in V for the ester substrate. The reaction of thrombin with 2-acetoxy-5-nitrobenzyl bromide, a substrate analogue, also resulted in the inactivation of the enzyme. The data are interpreted to show the presence of a tryptophan residue at or near the enzyme's substrate binding site.     

Chemical modification of the tryptophan residue in toxin B from the venom of the Indian cobra

The single tryptophan residue in toxin B has been converted into N′-formylkynurenine by ozonization in anhydrous formic acid, and also modified by reactions with 2-hydroxy-5-nitrobenzyl (HNB) bromide and 2-nitro-4-carboxyphenylsulphenyl (NCPS) chloride. Amino acid analyses of such modified derivatives show these reactions to be specific for tryptophan without significant effect to other amino acids. Ozonized toxin B has a residual toxicity of 80 %, and other tryptophan modified toxins retain at least half the toxicity of native toxin B. Each modified derivative gave a single fused precipitin line with native toxin on immunodiffusion against antitoxin B sera. In heterologous precipitin reactions, no significant decreases in antigenic activity of the modified derivatives were observed. The tryptophan residue at position 25 may, therefore, be part of neither the active site nor the antigenic site.     

Structure of 2-hydroxy-5-nitrobenzylated carboxypeptidase A.

Bovine pancreatic carboxypeptidase A (EC 3.4.12.2) was treated with dimethyl (2-hydroxy-5-nitrobenzyl)sulfonium chloride at pH 7.5, resulting in a preparation which consisted primarily of a monohydroxynitrobenzylated derivative of the enzyme. Samples of the hydroxynitrobenzylated enzyme were subjected to tryptic digestion and to cyanogen bromide cleavage, and resulting peptides were isolated chromatographically. One tryptic hydroxynitrobenzyl-containing peptide was isolated; its amino acid composition was that of the N-terminal tryptic segment of carboxypeptidase Agamma (residues 8--35). Likewise, CNBr cleavage of the hydroxynitrobenzylated enzyme revealed that the hydroxynitrobenzyl group resided in the N-terminal fragment, FN (residues 8--22). Neither of these hydroxynitrobenzylated peptides contains Trp, the amino acid residue which is characteristically the site of hydroxynitrobenzylation in proteins, and each was found to contain approximately one less Asx than the corresponding native peptide. Both dansylation and automated Edman degradation procedures revealed that the N-terminal Asn of carboxypeptidase Agamma had been modified by hydroxynitrobenzylation of the enzyme. Thus the sulfonium salt reacts with carboxypeptidase A in the same manner as that established earlier for 2-hydroxy-5-nitrobenzyl bromide (Radhakrishnan, T.M., Bradshaw, R.A., Deranleau, D.A. and Neurath, H. (1970) FEBS Lett. 7, 72--76). Such reactivity of the alpha-amino group presumably reflects its unique location with respect to Trp residues in the tertiary structure of the enzyme.     

The role of tryptophanyl residues in heavy meromyosin as studied by chemical modification with 2-hydroxy-5-nitrobenzyl bromide.

1. Two moles of 2-hydroxy-5-nitrobenzyl group bound selectively to one mole of heavy meromyosin when it was treated with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for tryptophanyl residues. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum with and without ADP of the bound 2-hydroxy-5-nitrobenzyl groups were measured with heavy meromyosin modified with various amounts of reagent. The properties of the modified heavy meromyosin did not change until the molar binding ratio of the reagent, rH, was about 1, but the properties changed remarkably when rH increased from 1 to 2. 2. Subfragment-1 was prepared from the modified heavy meromyosin by trypsin [EC 3.4.21.4] digestion. The molar binding ratio of the reagent in subfragment-1, rS, was found to be less than 0.1 when rH of the starting heavy meromyosin was less than 0.8. However, rS was about 0.5 in subfragment-1 prepared from heavy meromyosin of rH about 2. The results indicate that only one mole of 2-hydroxy-5-nitrobenzyl group, which was bound with lower reactivity than the other, was bound to a head part of heavy meromyosin. 3. Subfragment-1 fraction prepared from the modified heavy meromyosin could be separated into two fractions by DE-32 cellulose column chromatography; the subfragment-1 portion which eluted later showed a higher rS than that eluted in front. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum induced by ATP were measured with the modified subfragment-1 separated by DE-32 cellulose column chromatography. The ADP-binding ability and the size of the initial burst were not dependent on rS, and coincided with those of subfragment-1 prepared from unmodified heavy meromyosin. 4. The results of ADP binding studies suggest that heavy meromyosin is constituted from nonidentical subunits, and that there is an interaction between them which controls the ADP binding. Two tryptophanyl residues having specific reactivity toward 2-hydroxy-5-nitrobenzyl bromide are assumed to be involved in the interaction.     

Ultraviolet photoinactivation of galactosyltransferase. Protection by substrates.

Galactosyltransferase was irreversibly inactivated upon exposure to ultraviolet light and the rate of inactivation followed apparent first-order kinetics. Significant protection against inactivation was observed in the presence of various combinations of substrates. UDPgalactose and Mn2+ together gave the most protection. Amino acid analyses revealed the loss of 1 mol of tryptophan per mol of galactosyltransferase upon ultraviolet photoinactivation. Further evidence for an essential trypotphan was provided by difference spectra and by inactivation with 2-hydroxy-5-nitrobenzyl bromide and protection against this reagent by Mn2+ and UDPgalactose. The protection by UDPgalactose and Mn2+ was greater than that provided by UDPgalactose alone. Since Mn2+ provided no protection by itself, this suggested that the formation of the galactosyltransferase-Mn2+-UDPgalactose complex caused a conformational change which was responsible for the observed protection of the essential tryptophanyl residue.     

The role of tryptophan residues in heparin-antithrombin interactions

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of “heparin cofactor” activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent “activation” of the macromolecule.     

The lysine binding sites of human plasminogen. Evidence for a critical tryptophan in the binding site of kringle 4

Chemical modification of human degraded form of plasminogen with NH2-terminal lysine (Lys-plasminogen) and the elastase fragments kringle 1 + 2 + 3 and kringle 4 with the tryptophan reagent [14C]dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide results in the incorporation of label and the parallel loss of lysine binding ability. In the case of kringle 4, only one-half of the lysine binding sites could be inactivated, but the modified and unmodified forms could be separated by affinity chromatography. The modified form contained 1 mol of 2-hydroxy-5-nitrobenzyl groups/mol of kringle 4 and did not bind to lysine-Sepharose. Lysine analogs such as 6-aminohexanoic acid protected kringle 4 against modification. Peptide-mapping studies on this form showed that essentially all of the label was in two chymotryptic peptides containing a tryptophan corresponding to Trp426 in the plasminogen sequence. Competition experiments with anti-kringle 4 antibodies having an affinity for the lysine binding site showed that the binding of 2-hydroxy-5-nitrobenzyl-kringle 4 to antibodies was about 10 times weaker than for unmodified kringle 4. These results indicate that the integrity of specific tryptophan residue is critical to the binding of lysine and related amino acids to kringle 4of human plasminogen.     

Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site.

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.     

Chemical modification of rat liver arginase

Chemical modifications were used to search for catalytically important residues of rat liver arginase. The results of carbamoylation, nitration and diazotization suggest that lysyl and tyrosyl residues are not involved in the catalytic function of arginase. The modification of 5--6 tryptophanyl residues by N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide led to about 90% inhibition of the enzyme activity. Photooxidation of 21 histydyl residues also led to considerable inactivation of arginase. The modification of tryptophanyl and histidyl residues did not cause dissociation of the enzyme into subunits.     

The modification of the lone tryptophan residue in human serum albumin by 2-hydroxy-5-nitrobenzyl bromide. Characterization of the modified protein and the binding of L-tryptophan and benzodiazepines to the tryptophan-modified albumin.

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.     

Tryptophan modification by 2-hydroxy-5-nitrobenzyl bromide studied by MALDI-TOF mass spectrometry

The reaction with 2-hydroxy-5-nitrobenzyl bromide (HNB) is a common covalent modification of tryptophan. It results in several products which have been described by classical physico-chemical methods. To improve the understanding of the HNB-modified tryptophan structure, we synthesized a model peptide containing one tryptophan only, modified it by HNB, and analyzed the product by MALDI-TOF mass spectrometry. Surprisingly, several multi-modified products (up to 5 HNB moieties per one tryptophan) were identified. the influence of HNB concentration and pH on the degree of modification was also analyzed. In addition, a splitting of modified tryptophan peaks in MALDI-TOF spectrum was described; most probably, this effect is a common MALDI artifact of nitro-aromatic compounds which facilitates the identification of HNB-modified tryptophan by MALDI-TOF MS significantly.     

The chemistry of the reaction of 2-hydroxy-5-nitrobenzyl bromide with his-32 of alpha-lactalbumin.

His-32 of bovine or human alpha-lactalbumin reacts with the tryptophan reagent 2-hydroxy-5-nitrobenzyl bromide at pH 7. The reaction depends on the native conformation of the alpha-lactalbumin molecule and it is restricted to position 1 of the imidazole nucleus. The synthesis and characterization of 1-(2-hydroxy-5-nitrobenzyl)-histidine, 3-(2-hydroxy-5-nitrobenzyl)-histidine and 1,3-bis(2-hydroxy-5-nitrobenzyl)-histidine are described.     

Molecular dynamics simulations of helix folding: The effects of amino acid substitution

Molecular dynamics simulations were applied to helix folding of alanine-based synthetic peptides. A single alanine residue in the middle of the peptide was substituted with various nonpolar amino acids (leucine, isoleucine, valine, glycine or proline) to study the effect of the substitution. Unlike many other molecular dynamics simulations, nonhelical initial conformations were used in our simulations to study the folding process. An average solvent effect was included in the energy function to simplify the solvent calculation and to overcome the multiple minima problem. During the simulations, the peptides folded into helices in nanoseconds. Compact structures containing two helical segments were also observed. The calculated helical ratios of the peptides showed the same rank order as observed experimentally for the alanine-based peptides. Within a peptide, the helical ratio of each residue was calculated and a minimum was found near the center of the sequence for all peptides. The substitutions had different asymmetric effects on the helical ratios of the residues preceding and following the substitution site, indicating different helix capping preferences of the substituting amino acids. © 1997 John Wiley & Sons, Inc. Biopoly 42: 633–644, 1997     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Modification of myosin subfragment 1 tryptophans by dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide

Modification of tryptophanyl residues (Trps) of myosin subfragments 1 (S-1) was performed with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS). Under controlled conditions, pH 6 at 0 degrees C and 10-min reaction with 10-100-fold molar excess, K+(EDTA) activity was reduced down to less than half, whereas Ca2+-ATPase activity increased and acto-S-1-ATPase was not affected. The number of modified Trps (up to 2.5) agreed well with the number of 2-hydroxy-5-nitrobenzyl moieties incorporated in S-1. The thiol groups of S-1 were not affected up to 50-fold molar excess of DHNBS, thus indicating that the modification was selective for Trps. The modification of as few as one Trp caused a blue shift of the emission spectrum, accompanied by a reduction in the fluorescence quantum yield. The accessibility of Trps to the fluorescence quencher acrylamide is drastically reduced upon modification, indicating that DHNBS-reactive Trps are more "exposed" than the DHNBS-refractive ones. DHNBS modification did not seem to affect the ATP-induced tryptophan fluorescence enhancement of S-1. The effect of DHNBS modification of the intrinsic fluorescence of S-1 indicates that the modified Trps are located in a polar environment and that they may be identical with the long-lifetime Trps of Torgerson [Torgerson, P. (1984) Biochemistry 23, 3002-3007]. The most reactive Trp is located in the N-terminal 27-kDa fragment of the S-1 heavy chain. It might also be inferred from the above data that the nonexposed and ATP-perturbed Trp(s) is (are) located in the 50-kDa fragment.     

Is the subunit the minimal function unit of creatine kinase?

The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M'M') and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M'B), as isolated by PAGE, has the same Km for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB') has half the activity of the native muscle enzyme. The M'B, MB' and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M"B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide and the native brain enzyme submit. The above results suggest strongly the independent catalytic function of the subunit of creatine kinase.     

Modification of the neurotoxin RTX-III from the sea anemone Radianthus macrodactylus

The influence of chemical modification on neurotoxin RTX-III toxicity in mice has been studied. The toxicity was not affected by modification of Trp30 residue with 2-hydroxy-5-nitrobenzyl bromide but was diminished by a factor of 100 after reduction of the toxin's two disulfide bonds with 2-mercaptoethanol followed by derivatization with iodoacetamide. Blocking carboxyl groups with [3H]glycine methyl ester in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide led to only a two-fold drop in toxicity in the case of monocarboxylate-modified derivatives and a six-fold decrease for dimodified derivatives. A conception of multipoint attachment of the toxin to sodium channel is discussed.