Editor-in-Chief: Brüne, Bernhard
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Cloning, Purification and Characterisation of Cystathionine γ-Synthase from Nicotiana tabacum
Citation Information: Biological Chemistry. Volume 380, Issue 10, Pages 1237–1242, ISSN (Print) 1431-6730, DOI: 10.1515/BC.1999.157, June 2005
- Published Online:
Cystathionine γ-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine γ-synthase catalyses the pyridoxal 5′-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1 ± 3.1mM and of 0.23 ± 0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (K i = 18 ΜM, k inact = 0.56 min−1), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 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 K i values of 0.20, 0.30, 0.45, and 0.027mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine γ-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine γ-synthase diffracting to beyond 2.7 Å resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P212121 with unit cell constants a = 120.0 Å, b = 129.5 Å, c = 309.8 Å, corresponding to two tetramers per asymmetric unit.
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