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PLANT PHYSIOLOGY , Vol 104, Issue 4 1215-1220, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Purification and Properties of Cystathionine [gamma]-Synthase from Wheat (Triticum aestivum L.)
B. D. Kreft, A. Townsend, H. D. Pohlenz and B. Laber
Schering AG, Agrochemical Research, D-13342 Berlin, Germany
Cysthathionine [gamma]-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
cyanoboro[3H]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. The
pyridoxal 5[prime]-phosphate-dependent forward reaction has a pH optimum of
7.5 and follows a hybrid ping-pong mechanism with Km values of 3.6 mM and
0.5 mM for L-homoserine phosphate and L-cysteine, respectively. L-Cysteine
methyl ester, thioglycolate methyl ester, and sodium sulfide were also
utilized as thiol substrates. The 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
DL-propargylglycine (Ki = 45 [mu]M, Kinact = 0.16 min-1). Furthermore, the
homoserine phosphate analogs 4-(phosphonomethyl)-pyridine-2-carboxylic
acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and
DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive
inhibitors with Ki values of 45, 40, and 1.1 [mu]M, respectively.
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