Tryptophan hydrolase

A large and diverse group of proteins, including enzymes, cytoskeleton, contractile proteins, and receptors, have been shown to be modified by calpains. Thus, a number of enzymes such as tyrosine hydrolase, tryptophan hydrolase, transglutaminase, protein kinase C, and membrane Ca2+-ATPase are activated by calpain proteolysis [38]. Several receptor proteins, in particular receptors for steroid hormones, growth factors, and adrenaline, are modulated by calpains, which participate also in platelet activation, cell fusion, and mitosis [39], Although the physiological roles of calpains continue to be un-... 

Both enzymes belong to the family of a,p-hydrolases." The active site of MeHNL is located inside the protein and connected to the outside through a small channel, which is covered by the bulky amino acid tryptophane 128." It was possible to obtain the crystal structure of the complex with the natural substrate acetone cyanohydrin with the mutant SerSOAla of MeHNL. This complex allowed the determination of the mode of substrate binding in the active site." A summary of 3D structures of known HNLs was published recently." " ... 

Treatment of the enzyme with A-bromosuccinimide oxidized 2 of the 8 tryptophan residues and 8 of the 20 tyrosine residues, but none of the histidine residues. This treatment causes the phosphotransferase activity with tris as an acceptor to double and the hydrolase activity to increase slightly. In the case of cobalt alkaline phosphatase, the above treatment caused a threefold increase in hydrolase activity and the generation of an even greater phosphotransferase activity (91). 

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. 

A careful study of indolic compounds produced by R. phaseoli showed that tryptophan could be converted into lAA, indole-3-ethanol, and indole-3-methanol, but indole-3-acetamide was not formed [189]. Bradyrhizobium, however, were shown to contain indole-3-acetamide as well as the indoleacetamide hydrolase activity, suggesting the presence of the tryptophan monooxygenase/indoleacetamide hydrolase pathway [190,191] in these organisms. 

The experiments discussed above on the biosynthesis of a pseudan have also shown that kynurenic acid is not the precursor of the 2-alkylquinolin-4(lH)-ones. Based on the putative function of the genes of the qbs operons (176), a pathway was proposed for the biosynthesis of kynurenic acid (I), xanthurenic acid [10(8)], and quinolobactin [IO(8) j in Pseudomonas fluorescens ATCC 17400 (Scheme 5). The first step, the oxidation of tryptophan to N-formylkynurenine, is likely to be catalyzed by the enzyme tryptophan 2,3-dioxygenase (TDO) (QbsF), which is a heme-dependent enzyme. The second step, the deformylation of N-formyl-kynurenine to L-kynurenine is catalyzed by kynurenine formamidase (KFA). The product of qbsH, a metal-dependent hydrolase found also in other bacterial genomes, is the likely candidate. 

Finally, several other animal tissues yield useful enzymes that have been employed in S5mthesis. Pepsin is an important digestive protease from animal stomach whose native role is hydrolyzing amide bonds involving hydrophobic, aromatic amino adds, for example, phenylalanine, tyrosine, and tryptophan. Acylase from pordne kidney sdectivdy hydrolyzes N-acetyl amino adds and is commercially available. It has long been used for kinetic resolutions of amino adds. In addition to hydrolases, other animal enzymes have found important applications in biocatalysis. Rabbit musde aldolase is commerdally awiilable and was shown to catalyze aldol condensations between dihydroxyacetone phosphate and various nonnatural aldehydes by the Whitesides group in 1989 [10]. This seminal report touched off an avalanche of new applications for this and related enzymes in asymmetric synthesis. 

Selective hydrolysis of esters is a well-established procedure for the resolution of chiral carboxylic acids. Enzymes such as hydrolases, lipases and proteases are utilized. Due to their high selectivity for (5)-amino acids, proteases have been widely used in the selective transformations of amino acids and their derivatives. a-Chymotrypsin- a serine protease -catalyzes not only the hydrolysis of amide bonds, but also the cleavage of various esters, including a-alkyl-a-amino acid esters. One application is the synthesis of (5)-a-[ C]-methyltryptophan The anion synthesized by LDA-deprotonation of M-benzylidene tryptophan methyl ester (T) was alkylated with [ CJmethyl iodide to obtain the methyl A-benzylidene derivative 2, which was hydrolyzed under acidic conditions. Subsequent selective cleavage of the ester group with a-chymotrypsin provided the enantiomerically pure (5)-amino acid 3 in 33% radiochemical yield. 

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