Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans.
1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin  in a peptide "map". 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.
D000596:Amino Acids / Q000032:analysis; D001120:Arginine / Q000032:analysis; D004220:Disulfides / Q000032:analysis; D006836:Hydro-Lyases / Q000032:analysis*; D007656:Ketoglutaric Acids; D008239:Lysine / Q000032:analysis; D008968:Molecular Conformation; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011549:Pseudomonas / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D014461:Ultracentrifugation
D000596:Amino Acids; D004220:Disulfides; D007656:Ketoglutaric Acids; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D001120:Arginine; D006836:Hydro-Lyases; D008239:Lysine