Purification and Properties of Cystathionine y - Synthase f rom Wheat ( Triticum aestivum 1 . )
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چکیده
Cystathionine y-synthase (CS), an enzyme involved in methionine biosynthesis, was purified from an acetone powder prepared from wheat (Triticum aestivum L.). After several chromatographic steps and radiolabeling of the partially purified enzyme with sodium cyan~boro[~H]hydride, a single polypeptide with a molecular weight of 34,500 was isolated by sodium dodecyl sulfate-high performance electrophoresis chromatography. Since the molecular weight of the native enzyme was 155,000, CS apparently consists of four identical subunits. l h e pyridoxal 5'-phosphate-dependent forward reaction has a pH optimum of 7.5 and follows a hybrid ping-pong mechanism with K,,, values of 3.6 mM and 0.5 mM for Lhomoserine phosphate and i-cysteine, respedively. i-Cysteine methyl ester, thioglycolate methyl ester, and sodium sulfide were also utilized as thiol substrates. l h e latter observation suggests that CS and phosphohomoserine sulfhydrase might be a single enzyme. CS does not seem to be a regulatory enzyme but was irreversibly inhibited by m-propargylglycine (Ki = 45 &M, K i , = 0.16 min-'). Furthermore, the homoserine phosphate analogs 4-(phosphonomethyl)-pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-lyl)pyridine-2-carboxylic acid, and ~~-€-2-amino-5-phosphono-3pentenoic acid acted as reversible competitive inhibitors with Ki vahes of 45, 40, and 1.1 PM, respectively.
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