Tryptophan residues chemical modification

Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD5Q values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulOde bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 a subunits of an acetylcholine receptor that is composed of a2 6 subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modification studies the invariant tryptophan and tyrosine residues of post-synaptic neurotoxins were shown to be of a critical nature to the toxicity function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. 

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 ... 

From kinetic (57) and chemical modification (58) studies, Hurst et al. concluded that the catalytic residues in a cellulase from Aspergillus niger are a carboxylate anion (pKa 4.0-4.5) and a protonated carboxyl group (pKa 5.0-5.5) with tryptophan and dicarboxylic amino acid residues involved in substrate binding. 

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. 

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. 

Chymotrypm, probably the most thorou y studied enzyme because of its stability and availability, primarily catalyzes the hydrolysis of amide bonds of proteins and peptides adjacent to the carbonyl group of the aromatic L-amino acid residues of tryptophan, tyro e, and phenylalanine. Therefore, the hydrophobic interaction between the active site and substrate molecules is believed to make a mryor contribution to the stabflity of enzyme-substrate complexes. In fact, the X-ray data showed that the a-chymotryfsin molecule is an elipsoid (51x40x40 A) and that the active site is a hydrophobic cavity of 10—12 Ax5.5 —6.5 Ax3.5 —4.5 A (5). In chemical modification experiments, histidyl imidazole 2, and seryl hydroxyl 1 groups were found to be directly involved in the catal) ic process. The formation of acyl enzyme intermediates at the seryl residue was demonstrated by phyacal and chemical means. 

He XH, Wu M, Li SY, Chu YZ, Chen J, Liu LY. Chemical modification of tryptophan residues in superoxide dismutase from camellia pollen and its fluorescence spectrum. Chem Res Chin Univ 2005 21(5) 562-5. 

Peptides that contain amino acid residues that are prone to chemical modification (Bronstrup, 2004), such as methionine, tryptophan, or cysteine, should not be considered. 

Transketolase from Baker s yeast, easily split into TPP and apoenzyme, consists of 2 identical subunits of 70000 daltons. 2 TPP molecules are bound per mole of enzyme in a reversible manner. All coenzyme analogs tested turned out to be inhibitory to the enzymatic activity. A very high affinity was found for oxi-TPP and tetrahydro-TPP. There is evidence for a charge transfer complex between a tryptophan residue of apo-transketolase and TPP from fluorescence quenching and circular dichroism measurements. The chemical modification of the tryptophan residue at the coenzyme binding site supports this concept. The decrease of the magneto circular dichroism band at 292 nm after the reaction of apo-TK with the dimethyl (-2-hydroxi-5-nitrobenzyl) sulfonium chloride is regarded as direct evidence for the modification of a tryptophan residue. 

The chemical modification of hen egg-white lysozyme by AT-bromosuccinimide has been studied kinetically using the stopped-fiow method and previous results have been confirmed. One most rapidly reacting L-tryptophan residue (probably Trp-62) was clearly distinguished, by its rate of modification, from four other residues. The residue could be protected by ethylene glycol, chitin, 2-acetamido-2-deoxy-D-glucose, or chitotriose, but not by D-glucono-1,4-lactone. 

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