Clarence H. Suelter, Jean Wang, E. E. Snell
Jul 15, 1976
Citations
2
Influential Citations
63
Citations
Journal
FEBS Letters
Abstract
Tryptophanase, a pyridoxal-phosphate dependent enzyme, catalyzes the degradation of tryptophan to yield indole, pyruvate and ammonia. Many other /3-substituted amino acids or amino acid derivatives such as cysteine, S-alkylcysteines, cysteine sulfinic acid, S-benzylcysteine, serine,/3-chloroalanine, O-benzylserine and t~,fl-diaminopropionic acid also undergo t~,/3-elimination to yield pyruvate, ammonia and the/3 substituent (for a review see [1 ] ). The mechanism of tryptophanase-catalyzed reactions has been studied extensively [ 1 ] ; the enzyme has also been used for the quantitative estimation of tryptophan and of pyridoxal 5'-phosphate (pyridoxal-P) [I ,2], as an aid to classification of bacteria, and more recently for the synthesis of tryptophan or 5-hydroxytryptophan from pyruvate, ammonia and indole or 5-hydroxyindole [3,4]. Tryptophanase is usually assayed by following production of indole from tryptophan in end-point assays [2] or by coupling pyruvate formation to the oxidation of NADH in the presence of excess lactate dehydrogenase (LDH). Since indole strongly inhibits, these assays are linear only for a short time unless indole is removed from solution during assay [2]. The coupled assay also requires additional enzymes and reagents. This paper describes a convenient direct spectrophotometric assay of this enzyme that uses a new, highly reactive chromogenic substrate, S-o-nitrophenyl-L-cysteine. 2. Materials and methods