Tryptophan residues catalytic properties

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 /8 subunit of tryptophan synthase from E. coli is inactivated by a mutation at Gly-281 (G281R).108 Gly-281 is located at a sharp turn in the trypsin-sensitive, subdomain (residues 260-310) that makes several contacts with the a subunit.7) The G281R mutation alters the catalytic properties of the isolated /8 subunit and weakens association with the a subunit. The mutation may interfere with hydrophobic interactions between the N-terminal and C-terminal domains of the /8 subunit and prevent a conformational change that affects catalytic properties and subunit interaction.108 Insertion of arginine or tryptophan between tyrosine 279 and phenylalanine 280 of the /3 subunit greatly weakens subunit interaction and decreases catalytic activity (X.-J. Yang and E.W. Miles, unpublished results). 

Finally, it could also be demonstrated that it is possible to improve VP catalytic properties by modifying the amino acid residues around the catalytic tryptophan. In this sense, simultaneous removal of Arg257 and incorporation of a phenylalanine residue, homologous to LiP Phe267 involved in VA binding [80], resulted in a VP variant with VA transient-state kinetic constants comparable to those of LiP [76]. 

Tire tetrahydrobiopterin formed in this reaction is similar in structure to a reduced flavin. The mechanism of its interaction with 02 could reasonably be the same as that of 4-hydroxybenzoate hydroxylase. However, phenylalanine hydroxylase, which catalyzes the formation of tyrosine (Eq. 18-45), a dimer of 451-residue subunits, contains one Fe per subunit,113 313i whereas flavin monooxygenases are devoid of iron. Tyrosine hydroxylase416 193 and tryptophan hydroxylase420 have very similar properties. All three enzymes contain regulatory, catalytic, and tetramerization domains as well as a common Fe-binding motif in their active sites.413 421 4213... 

Considerable information is available on the molecular properties of this enzyme, on the catalytic mechanism, and on the stereochemistry of the enzymic reaction. The isomerase is composed of identical associating subunits. The primary structure is known and comprises 125 residues (MW 13,394) including all the common amino acids except cysteine and tryptophan. The enzyme, which exhibits exceptionally high catalytic activity, has a molecular activity at saturating concentrations of A -androstene-3,17-dione of 4.38 X 10 min per monomer at pH 7.0 and 25°. Mechanistic studies have disclosed that the isomerase catalyzes the reaction of A < -3-ketostcroids by a direct, stercospccific, and intramolecular transfer of the 4 8-proton to the 6)8 position. There is considerable evidence for the involvement of an enolic intermediate in the... 

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