A colorimetric assay for a pyridoxal phosphate-dependent β-replacement reaction with l-cysteine: Application to studies of wild-type and mutant tryptophan synthase α2β2 complexes
✍ Scribed by Arvind M. Kayastha; Edith Wilson Miles
- Publisher
- Elsevier Science
- Year
- 1991
- Tongue
- English
- Weight
- 428 KB
- Volume
- 193
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.