|
type |
Journal Article |
authors |
Ravanel, S.; Gakiere, B.; Job, D.; Douce, R. |
title |
Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli |
journal |
Biochem J |
Activity |
2.5.1.48 |
Family |
2.5.1.48.b |
sel |
selected |
ui |
9531508 |
year |
(1998) |
volume |
331 |
number |
Pt 2 |
pages |
639-48 |
| |
keywords |
Alkynes/pharmacology |
abstract |
Cystathionine gamma-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine gamma-synthase from Arabidopsis thaliana has been cloned and used to overexpress the enzyme in Escherichia coli. The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5'-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 microM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine gamma-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint gamma-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the gamma-replacement reaction and approx. 1/1500 as fast as the gamma- elimination catalysed by the E. coli enzyme. This huge difference could be attributed to the inability of the A. thaliana cystathionine gamma- synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient gamma-elimination reaction catalysed by the enterobacterial enzyme. |
last changed |
2009/02/11 18:24 |
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