Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase.
High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D050960:Aminomethyltransferase; D002352:Carrier Proteins / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000378:metabolism*; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism; D019877:Hydroxymethyl and Formyl Transferases; D008928:Mitochondria / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D013763:Tetrahydrofolates / Q000096:biosynthesis / Q000494:pharmacology; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D014166:Transferases / Q000378:metabolism
D002352:Carrier Proteins; D050961:Glycine Decarboxylase Complex H-Protein; D009097:Multienzyme Complexes; D010940:Plant Proteins; D011768:Pyruvate Dehydrogenase Complex; D013763:Tetrahydrofolates; C064525:glycine cleavage system; C013123:5,10-methylenetetrahydrofolic acid; D009243:NAD; C030371:5,6,7,8-tetrahydrofolic acid; D008063:Thioctic Acid; C015144:dihydrolipoic acid; D000594:Amino Acid Oxidoreductases; D050841:Glycine Dehydrogenase (Decarboxylating); D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D012696:Glycine Hydroxymethyltransferase; D050960:Aminomethyltransferase; D005998:Glycine