Cloning, Purification and Characterisation of Cystathionine γ-Synthase from Nicotiana tabacum
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Tim Clausen
Abstract
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 (Ki = 18 ΜM, kinact = 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 Ki 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.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Editors Note
- Walter Neupert: Spellbound by Mitochondria
- Posttranslational Protein Translocation Across the Membrane of the Endoplasmic Reticulum
- Import of Carrier Proteins into Mitochondria
- The Essential Role of Mitochondria in the Biogenesis of Cellular Iron-Sulfur Proteins
- Mitochondrial Iron Metabolism in the Yeast Saccharomyces cerevisiae
- A Scj1p Homolog and Folding Catalysts Present in Dog Pancreas Microsomes
- The Import Pathway of Human and Thermoplasma 20S Proteasomes into HeLa Cell Nuclei Is Different from That of Classical NLS-Bearing Proteins
- Role of p52 (NF-kappaB2) in LPS Tolerance in a Human B Cell Line
- SHP1 Protein Tyrosine Phosphatase Negatively Modulates Erythroid Differentiation and Suppression of Apoptosis in J2E Erythroleukemic Cells
- Comparative Cleavage Sites within the Reactive-Site Loop of Native and Oxidized α1-Proteinase Inhibitor by Selected Bacterial Proteinases
- A High Affinity Binding Site for the Polypyrimidine Tract Binding Protein (PTB) Is Located in the 5'-Untranslated Region of the Rat Proteinase α1-Inhibitor 3 Variant I Gene
- Defining the Location and Function of Domains of McrB by Deletion Mutagenesis
- Disruption of the Gene for Hsp30, an α-Crystallin-Related Heat Shock Protein of Neurospora crassa, Causes Defects in Import of Proteins into Mitochondria
- Cloning, Purification and Characterisation of Cystathionine γ-Synthase from Nicotiana tabacum
- Isolation and Characterization of Viridin, a New 65 kDa Antifungal Protein from the Mould Trichoderma viride
- The T-Knot Motif Revisited
