DYE-AFFINITY LABELING OF BOVINE HEART MITOCHONDRIAL MALATE-DEHYDROGENASE AND STUDY OF THE NADH-BINDING SITE

The ability of the reactive dichlorotriazine dye Vilmafix Blue AR (VBA R) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k (3) 0.16 min(-1); K-D 14.4 mu M). The inactivation of the NADH-depende nt enzyme by VBAR is competitively inhibited by NAD(+), NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be re covered either on incubation with 10 mM NAD(+), 10 mM NADH or 10 mM AD P, or by extensive dialysis or gelfiltration chromatography. Data obta ined from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by rev erse-phase HPLC analysis revealed one VBAR-labelled peptide. It appear s that each subunit features the same peptide bearing the modifying re sidue involved in MDH labelling. The pK(a) of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPL C and TLC analysis, and molecular-modelling studies suggest that the m odifying residue is Lys-81 and/or Lys-217.