Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria.
The aim of the present work was to further determine how the T-protein of the glycine-cleavage system and serine hydroxy-methyltransferase (SHMT), two folate-dependent enzymes from pea leaf mitochondria, interact through a common pool of tetrahydrofolate polyglutamates (H4PteGlun). It was observed that the binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. It was also established that, once bound to the proteins, tetrahydrofolate, a very O2-sensitive molecule, was protected from oxidative degradation. The dissociation constants (Kd) of H4PteGlu5, the most predominant form of polyglutamate in the mitochondria, were approximately 0.5 microM for both T-protein and SHMT, whereas the Kd values of CH2-H4PteGlu5 were higher, 2.7 and 7 microM respectively. In a matrix extract from pea leaf mitochondria, the maximal activity of the glycine-cleavage system was about 2.5 times higher than the maximal activity of SHMT. This resulted in a permanent disequilibrium of the SHMT-catalysed reaction which was therefore driven toward the production of serine and H4PteGlun, the thermodynamically unfavourable direction. Indeed, measurements of the steady-state ratio of CH2-H4PteGlun/H4PteGlun (n = 1 or n = 5) during the course of glycine oxidation demonstrated that the methylene form accounted for 65-80% of the folate pool. This indicates that, in our in vitro experiments, CH2-H4PteGlun with long polyglutamate chains accumulated in the bulk medium. This observation suggests that, in these in vitro experiments at least, there was no channelling of CH2-H4PteGlu5 between the T-protein and SHMT.
Rebeille
F
F
CEA, Physiologie Cellulaire Végétale, CNRS, Grenoble, France.
Neuburger
M
M
Douce
R
R
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D007887:Fabaceae / Q000201:enzymology / Q000378:metabolism*; D050841:Glycine Dehydrogenase (Decarboxylating); D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D010946:Plants, Medicinal; D011099:Polyglutamic Acid / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism*
D013763:Tetrahydrofolates; D011099:Polyglutamic Acid; D000594:Amino Acid Oxidoreductases; D050841:Glycine Dehydrogenase (Decarboxylating); D012696:Glycine Hydroxymethyltransferase