Tryptophan residues, modification

Teng LR, Chu YZ, Zhang XP, Wang J, Han S, Yu XK, et al. Studies on tryptophan residue modification and fluorescence spectrum of hyaluronidase. Chem J Chin Univ 2005 26(9) 1662-4. 

The one residue most extensively studied is tryptophan. It is very easily modified, indicating that tryptophan residue is exposed 5-8). Raman spectroscopic analysis of a sea snake neurotoxin indicated that a single tryptophan residue is indeed exposed (2). The tryptophan residue lies in the important loop consisting of segment 4. Modification of the tryptophan residue induces the loss of AChR binding ability as well as the loss of toxicity 5-8). 

The six major proteins of milk, asl-, o s2-, and /c-casein, jS-lactoglobulin, and a-lactalbumin, contain at least one tryptophan residue [57], the fluorescence of which allows the monitoring of the structural modifications of proteins and their physicochemical environment during the coagulation processes. Emission fluorescence spectra of the protein tryptophanyl residues were recorded for the milk coagulation kinetics induced by... 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

A previous study of the reaction of ozone with lysoz3mie dissolved in anhydrous formic acid gave rise to the conclusion that the only amino acid residues affected in the early stages of the reaction were the tryptophan residues 108 and 111 ( ). Conversion of these residues to N -formyl-kynurenine did not cause loss in enzyme activity. Imoto et al. (7) have pointed out that this result is anomalous since modifications of tryptophan 108 (e.g. with iodine) normally causes inactivation. 

Modification of Tryptophan Residues. Tryptophan residue also could be determined quantitatively by a modification with a sulfenylating agent such as 2-nitrophenyl sulfenyl chloride in 30% acetic acid ( ). Since both oxidized and sulfenylated tryptophan gave characteristic absorption at 365 nm, the extent of tryptophan modification was calculated from the mean difference between the sulfenylated protein and the kynurenine. The absorption contributed by kynurenine was comparatively weak. 

The chemical synthesis of the Amanita toxins has presented several problems, in particular those related to the formation of the sulfur bridge. The latter has been explored with model compounds.[2 31 It has been found that the synthesis of the (sulfanyl)indole moiety can be achieved by reacting an indole compound with an alkanesulfenyl chloride. A model tryptathionine compound has been prepared by reacting A-acyl-L-cysteine and /V-acyl-L-tryptophan in the presence of A-chlorosuccinimide in glacial acetic acid at room temperature.[4] The sulfanylation reaction has been subsequently exploited for the selective chemical modification of tryptophan residues in proteins using 2-nitrophenylsulfenyl chlorideJ5 ... 

The single trytophan residue in the native RNase Tt seems to be embedded in the interior of the molecule and cannot be modified by specific reagents. The modification of the residue in 8M urea was carried out with 2-hydroxyl-5-nitrobenzyl bromide by Takahashi (64a) and with A-bromosuccinimide by Kawashima and Ando (65). In both cases complete inactivation occurred when the tryptophan residue was completely modified. However, RNase T, modified with 2-hydroxyl-5-nitrobenzyl bromide in 4 M urea retained about 30% of the original activity (66). Thus, it may be concluded that the tryptophan residue does not participate directly in the catalytic process, since it is situated quite close to the essential Glu 58, it may indirectly participate in the building up of the active conformation (11). 

Oxidation of two out of 13 tryptophan residues in a cellulase from Penicillium notatum resulted in a complete loss of enzymic activity (59). There was an interaction between cellobiose and tryptophan residues in the enzyme. Participation of histidine residues is also suspected in the catalytic mechanism since diazonium-l-H-tetrazole inactivated the enzyme. A xylanase from Trametes hirsuta was inactivated by N-bromosuc-cinimide and partially inactivated by N-acetylimidazole (60), indicating the possible involvement of tryptophan and tyrosine residues in the active site. As with many chemical modification experiments, it is not possible to state definitively that certain residues are involved in the active site since inactivation might be caused by conformational changes in the enzyme molecule produced by the change in properties of residues distant from the active site. However, from a summary of the available evidence it appears that, for many / -(l- 4) glycoside hydrolases, acidic and aromatic amino acid residues are involved in the catalytic site, probably at the active and binding sites, respectively. 

F. Yamakura et al., Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species. Nitric Oxide 14, 152-161 (2006)... 

The chemical modifications of the tryptophan residues lead to a decrease in the nutritive value of proteins as observed in autoclaved soja meals (124), heated meats (125), heated casein (126), and heated skim milk (122) this last reference is probably the most reliable work published in this field. The nutritional effects and the metabolic transit of heat-treated and oxidized tripeptide (gly-try—gly) have been investigated (123,132,137) recently only the metabolic transit study is related here. 

The comQXP loci of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum sensing response.217 Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the posttranslational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various phenotypes. 

The other amino acid residue present in proteins that is susceptible to oxidation is the indole moiety of tryptophan (Fig. 11). The reducing potential of tryptophan is considerably less than that of cysteine and methionine, so oxidation of tryptophanyl residues usually does not occur until all exposed thiol residues are oxidized. Also, the spontaneous oxidation of tryptophanyl residues in proteins is much less probable than that of cysteinyl and methionyl residues. Tryptophan residues are the only chromophoric moieties in proteins which can be photooxi-dized to tryptophanyl radicals by solar UV radiation, even by wavelengths as long as 305 nm (B12). Tryptophanyl residues readily react with all reactive oxygen species, hypochlorite, peroxynitrite, and chloramines. Oxidative modifications of other amino acid residues require use of strong oxidants, which eventually are produced in the cells. Detailed mechanisms of action of these oxidants is described in subsequent sections of this chapter. 

K4. Kato, Y., Kawakishi, S., Aoki, T., Itakura, K., and Osawa, T., Oxidative modification of tryptophan residues exposed to peroxynitrite. Biochem. Biophys. Res. Commun. 234, 82-84 (1997). 

The oxidation of trypsin and trypsinogen was carried out in aqueous 0.1 M acetate buffer solutions at room temperature. In this particular case and under these conditions no significant cleavage of peptide bonds next to tryptophan residues occurred. Careful analysis of hydrolyzates of NBS-oxidized trypsinogen and trypsin confirmed the selectivity of the oxidative modification of the protein, as Table XXIV shows. There is no significant loss of tyrosine, histidine, serine, threonine, or cystine, although all of these amino acids will react with NBS but considerably less rapidly than tryptophan. 

In the absence of efficient Nps-Q (92) capture, this intermediate reacts with the indole side chain of tryptophan residues to produce the related 2-Nps derivatives 96 (Scheme 48). Although reaction of sulfenyl hahdes with indoles was exploited by Wieland et al.t for the synthesis of phaUoidin, this side reaction leads to irreversible modification of Trp-containing peptides.This side reaction does not occur via an intramolecular electrophilic substitution as postulated previously,but by a direct attack of the Nps-Cl (92) in fact, it is efficiently suppressed by the addition of a large excess of an indole derivative as a scavenger.These scavengers serve also to decrease the proton activity of the acids vide supra). Due to the unpleasant odor of 2-methylindole the less volatile 1-acetyltryptophan butyl ester has been proposed as scavenger.f ... 

The procedure outlined below, based upon the description of Previero et al. (1967b) has been used successfully, with minor variations for the selective modification of tryptophan residues in lysozyme (Previero et al. 1967b), trypsin (Coletti-Previero et al. 1969), cytochrome c (Aviram and Schejter 1971), and thioredoxin (Holmgren 1972). 

Site-specific sulfenylation of tryptophan residues in egg-white lysozyme has been attained by either limiting the amount of reagent, or modifying the reaction conditions. Thus, Trp-108 is the major site of modification upon addition of one equivalent of 2-thio-(2-nitro-4-carboxyphenyljsulfenyl chloride to lysozyme in 25% acetic acid (Veronese et al. 1972). Specific modification of Trp-62 was attained in approximately 8 hr by the addition of 5 consecutive portions of solid NPS-Cl (10 /imole for each ml of reaction mixture) to a solution of lysozyme (0.5 /imole/ml) in 0.1 M sodium acetate at pH 3.5 (Shechter et al. 1972). In both cases, the protein derivative was separated from other products, and unreacted enzyme by ion-exchange chromatography. 

---