A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes.
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.
Kayastha
A M
AM
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Miles
E W
EW
D003124:Colorimetry / Q000379:methods; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D008751:Methylene Blue / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010655:Phenylenediamines / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D012486:Salmonella typhimurium / Q000201:enzymology; D013440:Sulfides / Q000737:chemistry; D014367:Tryptophan Synthase / Q000737:chemistry
D010655:Phenylenediamines; D013440:Sulfides; D011732:Pyridoxal Phosphate; C004781:dimethyl-4-phenylenediamine; D014367:Tryptophan Synthase; D003545:Cysteine; D008751:Methylene Blue