Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii.
An isocitrate dehydrogenase able to function with either NADP or NAD as coenzyme was purified to homogeneity from cell-free extracts of the purple photosynthetic eubacterium Rhodomicrobium vannielii using a rapid two-step procedure involving dye-ligand affinity chromatography. The enzyme was obtained in 60% yield with specific activities of 23 U.mg protein-1 (NADP-linked reaction) and 18.5 U.mg protein-1 (NAD-linked reaction). The purified enzyme was monomeric and migrated with an approximate Mr of 75,000-80,000 on both SDS/PAGE and non-denaturing PAGE. Affinity constants (Km values) of 2.5 microM for NADP and 0.77 mM for NAD and values for kcat/Km of 981,200 min-1.mM-1 (NADP) and 2455 min-1.mM-1 (NAD) indicated a greater specificity for NADP compared to NAD. A number of metabolites were examined for possible differential regulatory effects on the NADP- and NAD-linked reactions, using a dual-wavelength assay. Oxaloacetate was found to be an effective inhibitor of both reactions and the enzyme was also sensitive to concerted inhibition by glyoxylate and oxaloacetate. The amino-acid composition and the identity of 39 residues at the N-terminus were determined and compared to other isocitrate dehydrogenases. The results suggested a relationship between the Rm. vannielii enzyme and the monomeric isocitrate dehydrogenase isoenzyme II from Vibrio ABE-1.
Leyland
M L
ML
Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, England.
Kelly
D J
DJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010071:Oxaloacetates / Q000494:pharmacology; D012244:Rhodospirillaceae / Q000201:enzymology*
D000596:Amino Acids; D010071:Oxaloacetates; D009243:NAD; D009249:NADP; D007521:Isocitrate Dehydrogenase