Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii

Eur J Biochem. 1991 Nov 15;202(1):85-93. doi: 10.1111/j.1432-1033.1991.tb16347.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Chromatography, Affinity
  • Hydrogen-Ion Concentration
  • Immunoblotting
  • Isocitrate Dehydrogenase / chemistry
  • Isocitrate Dehydrogenase / isolation & purification*
  • Isocitrate Dehydrogenase / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • NAD / metabolism
  • NAD / pharmacology
  • NADP / metabolism
  • NADP / pharmacology
  • Oxaloacetates / pharmacology
  • Rhodospirillaceae / enzymology*

Substances

  • Amino Acids
  • Oxaloacetates
  • NAD
  • NADP
  • Isocitrate Dehydrogenase

Associated data

  • GENBANK/UNKNOWN