Abstract
Abstract Cysteine synthetase in Salmonella typhimurium is a multifunctional protein complex of molecular weight 309,000 which catalyzes the two-step synthesis of l-cysteine from l-serine, acetyl-CoA, and sulfide. Certain enzymic and chemical properties of a purified preparation of cysteine synthetase have been studied. O-Acetyl-l-serine at a concentration of 10-4 to 10-3 m causes this complex to dissociate reversibly into 1 molecule of serine transacetylase (mol wt 160,000) and 2 molecules of O-acetylserine sulfhydrylase (mol wt 68,000). Serine transacetylase and O-acetylserine sulfhydrylase have been resolved from one another by Sephadex gel filtration in the presence of O-acetyl-l-serine. The O-acetylserine sulfhydrylase associated with serine transacetylase in the complex appears to be identical with the previously described free O-acetyl-serine sulfhydrylase.
Highlights
The 0-acetylserine sulfhydrylase associated with serine transacetylase in the complex appears to be identical with the previously described free O-acetylserine sulfhydrylase
The biosynthesis of L-cysteine in Salmonella typhimurium has previously been shown to proceed via a two-step pathway wherein L-serine is first acetylated by acetyl-CoA to yield O-acetyl-nserine; this reaction is catalyzed by serine transacetylase and is feedback-inhibited by L-cysteine (1). 0-Acetyl-L-serine reacts with sulfide in a reaction catalyzed by 0-acetylserine sulfhydrylase to form L-cysteine
For assays done in the presence of thiols, the same assay mixture was used, but DNB was omitted, and the reaction was followed by the loss of absorbance at 232 rnp which occurs with the cleavage of the thiolester bond of acetyl-CoA (1,3)
Summary
Materials-DNB1 was purchased from Aldrich, histidinol hydrochloride from Cycle Chemicals, and bovine serum albumin from Armour. Research and Development, Ltd. Pyridoxal phosphate and trypsin, Grade B, were obtained from Calbiochem. Acetyl-CoA, and silica gel thin layer chromatography plates were purchased from Mann, calcium phosphate gel from Sigma, and protamine sulfate from Nutritional Biochemicals. Other chemicals were obtained as previously described (2). Enzyme Assuys-Serine transacetylase was assayed spectrophotometrically by measuring the absorbance of the chromophore produced by the reaction of DNB with free coenzyme A, as described previously (1). For assays done in the presence of thiols (which would react with the DNB), the same assay mixture was used, but DNB was omitted, and the reaction was followed by the loss of absorbance at 232 rnp which occurs with the cleavage of the thiolester bond of acetyl-CoA (1,3)
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