Tryptophan residues replacement

Several deadly species of the genus Amanita produce colorless toxic octapeptides, the amani-tins.a b Two residues of glycine, one of L-isoleucine, one of the unusual L-dihydroxyisoleucine, one of L-asparagine, and one of L-hydroxyproline are present in a-amanitin. In the center a modified tryptophan residue has been combined oxidatively with an SH group of a cysteine residue. If the dihy-droxyisoleucine residue of a-amanitin is replaced with unhydroxylated leucine, the resulting compound, known as amanullin, is nontoxic. The LD50 for mice is 0.3 mg kg 1 and 50 g of fresh Amanita phalloides may be sufficient to kill a person. Arnan-itins act slowly, and it is impossible to kill mice in less than 15 h, no matter how high the dose. 

All lipases of this family are characterized by a helical lid that covers the active site (Kg. 9X It has been hypothesized [145] that this lid moves away in a hydrophobic environment, thereby making the active site accessible to the lipid substrate. In this hypothesis an important role has been reserved for the bulky tryptophane residue at position 117 in the center of the helical lid. In the Rhizoptts javwicus lipase, however, this bulky hydrophobic residue is replaced by a alanine, and no other bulky residue seems located so that it could take die role of residue 117. Therefore, it was concluded that the lipid-interaction model that haa been suggested for this class of lipase is not generally applicable in all its details. 

The emission peak at 500 nm was absent when the apoenzyme was excited at 290 nm47 (Fig. 9.11). This peak was also diminished in a mutant holoenzyme in which Trp-248 was replaced by phenylalanine.I8 It was proposed that this peak is due to energy transfer from the tryptophan residues to the coenzyme. Binding of anthranilic acid or alanine quenches the 500-nm peak and, therefore, interferes with the energy transfer.43 ... 

Structure-function roles have been suggested for unique tryptophan residues in other copper proteins as well (44,45, 46). Moreover, the single tryptophan that is quenched by including the copper atom in azurin is apparently not in contact with the indole ring, as evidenced by metal replacement and phosphorescence results (45, 46). 

Figure 9.6. Cation-71 interaction in the binding of acetylcholine to the nicotinic acetylcholine receptor, a Incorporation of flu-orinated tryptophan residues into NAR a chains in vivo. Individual tryptophan codons were replaced by amber stop codons (UAA). The mRNA was obtained by transcription in vitro and injected into Xenopus laevis (frog) oocytes, along with a suppressor tRNA that had been acylated with the synthetic fluorinated trp. b Oocytes expressing the modified NAR were analyzed by patch clamp. Currents are plotted as functions of acetylcholine concentration. The EC q increases monotonously with the extent of fluorination. c Replot of the EC q as a function of a derived parameter that describes the strength of the cation-71 interaction (the derivation is beyond me).

Recently, 2-(2-nitrophenylsulfenyI)-3-methyl-3-bromoindolenine (BNPS-skatole) has replaced N-bromosuccinimide, which had been used frequently in earlier studies. At low reagent to protein tryptophan ratios, in 50% aqueous acetic acid, BNPS-skatole reacts selectively with tryptophan residues converting these to the oxindole derivative. Methionine is concommitantly converted to the sulfoxide. At high concentrations of reagent, slow selective cleavage (to the extent of 15-60%) of the peptide bonds involving tryptophanyl residues is... 

Replacement of Cys-430 of the E. coH W aspartase gene with a tryptophan residue has been shown to enhance aspartase activity threefold at pH 6.0, but only slightly at pH 8, the optimum for the wild-type enzyme [40]. Limited proteolysis of the mutated protein completely inactivates the enzyme, showing that truncation activation and the Cys-430 mutation are not additive. Substitution of Lys-126 to an arginine residue of the E. coH AS. 881 aspartase causes a fivefold enhancement in specific activity at pH 8.5 with a concomitant increase in the thermostability... 

Figure 4-6. (A) A close-up view of the active site of yeast cytochrome c peroxidase showing the residues in the distal pocket at which hydrogen peroxide is reduced to water. Overlaid on the structure of the wild type enzyme are the positions of residues in the W51F mutant (tryptophan is replaced by phenylalanine). (B) Voltammograms of a film of wild type CcP on a PGE electrode, obtained in the absence and presence of H2O2 at ice temperature, pH 5.0. The electrode is rotating at 200 rpm, but the catalytic current in this case continues to increase as the rotation rate is increased therefore under these conditions the electrocatalysis is diffusion controlled and few facts are revealed about the enzyme s chemistry. For the W51F mutant, the signal due to the reversible two-electron couple and the catalytic wave are both shifted >100 mV more positive in potential compared to the wild-type enzyme. Reproduced from ref. 46 and 47 with permission.

In a multi-tryptophan protein, fluorescence quantum yield of the tryptophans can be additive or not. In the first case, the others do not influence each tryptophan while in the second case mainly energy transfer to or from the other tryptophan residues can influence quantum yield. In the absence of energy transfer between the tryptophan residues, mutants with one tryptophan residue are prepared, the other tryptophan residues of the protein are replaced by phenylalanine, and quantum yield of the corresponding tryptophan in each mutant is determined experimentally by comparing its fluorescence to that of free tryptophan in solution. For example, quantum yields of tryptophan residues in Bamase have been determined in the wild type and in mutants where only one tryptophan residue has been conserved. 

Troponin C typically lacks tryptophan residues. This is fortunate because it allows insertion of trypu han donors at any desired location by site-directed mutagenesis. In the mutant shown in Figure 14.15, a single tryptophan residue was placed at position 22 to serve as the donor. As is typical in the creation of mutant proteins, the tryptophan (W) was a conservative replacement for phenylalanine (F). A uniquely reactive site for the acceptor was provided by rqilacing asparagine-52 with a cysteine residue (N52C). This site was labeled witii lAEDANS. 

Small G-proteins contain two regions named switch I and switch II, which undergo a binary conformational change upon nucleotide exchange and GTP hydrolysis (Vetter and Wittinghofer, 2001). The switch II region of Arf and Sar contains a conserved tryptophan residue, which acts as an intrinsic fluorescent probe of the protein conformation. The intrinsic fluorescence of Arfl and Sari increases (by +100% and +200%, respectively) when GDP is replaced by GTP. Tryptophan fluorescence is thus a convenient way to follow the activation-inactivation cycle of these small G-proteins in real-time (Antonny et al, 1997,2001 Bigay et al., 2003 Futai et al, 2004). 

The replacement of Val and Gly by Ala and of Val by lie corresponds to the general scheme. No replacements of Ionized amino acids> however, can be observed. The drawback of all these methods Is that they are statistical In nature they Include therefore also the genetic drift which Is of more or less neutral character. A change In thermostability can be caused, however, by the replacement of a single amino acid, resulting, for example. In the formation of a new Ion pair [22] or In an aromatic-aromatic Interaction [23]. The latter type of stabilization can easily play a role In thermltase which, unlike subtlllsln BPN, contains four additional tyrosine resldued and three additional tryptophan residues. The final answer, however, will have to await the experimental determination of the three-dimensional structure of the enzyme. 

Bronskill, P.M., Wong, J.T. Suppression of fluorescence of tryptophan residues in proteins by replacement with 4-fluorotryptophan. Biochem. J. 249, 305-308 (1988)... 

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