Glycine decarboxylase multienzyme complex. Purification and partial characterization from pea leaf mitochondria.
The P, H, and T proteins of the glycine cleavage system have been purified separately from pea leaf mitochondria and demonstrate molecular weights of 98,000, 15,500, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of P protein by gel filtration was 210,000, indicating that this enzyme has a native homodimer conformation. Reconstitution assays containing purified P, H, and T proteins and yeast lipoamide dehydrogenase catalyze the oxidation of glycine and demonstrate a strict dependence on pyridoxal phosphate, tetrahydrofolate, NAD+, and dithiothreitol. The released CO2, methylamine-H protein intermediate, and methylenetetrahydrofolate are produced in stoichiometric amounts from glycine during the cleavage reaction. H protein acts as co-substrate with glycine during the decarboxylation reaction, demonstrating an apparent Km value of 2.2 microM. P and H protein alone jointly catalyze the glycine carboxyl-14 CO2 exchange reaction in the presence of pyridoxal phosphate and dithiothreitol. L protein of the glycine cleavage system was immunopurified using monoclonal antibodies. Antigenic and molecular weight similarities of the L protein with the lipoamide dehydrogenase component of the pyruvate dehydrogenase complex were shown suggesting the possibility of common isomers of lipoamide dehydrogenase for the two enzyme complexes.
Walker
J L
JL
Oliver
D J
DJ
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification*; D050960:Aminomethyltransferase; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000032:analysis* / Q000302:isolation & purification*; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification*; D010946:Plants, Medicinal; D014166:Transferases / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D001639:Bicarbonates; D002352:Carrier Proteins; D050961:Glycine Decarboxylase Complex H-Protein; D009097:Multienzyme Complexes; D010940:Plant Proteins; C064525:glycine cleavage system; D002245:Carbon Dioxide; D000594:Amino Acid Oxidoreductases; D050841:Glycine Dehydrogenase (Decarboxylating); D008058:Dihydrolipoamide Dehydrogenase; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase; D005998:Glycine