THE INTERACTION OF CANDIDA-BOIDINII FORMATE DEHYDROGENASE WITH A NEW FAMILY OF CHIMERIC BIOMIMETIC DYE-LIGANDS

Seven chimeric biomimetic dye-ligands (BM) are purpose-designed and sy nthesized by specific structural modification of the parent anthraquin one dichlorotriazine dye Vilmafix blue A-R (VBAR). Each BM dye is comp osed of two enzyme-recognition moieties. The terminal biomimetic moiet y bears a variable carboxylated structure linked to the triazine ring, thus mimicking the substrate of formate dehydrogenase (FDH). The anth raquinone moiety remains the same as that of the parent dye and recogn izes the nucleotide-binding area of the target enzyme, Dyes are purifi ed by liquid column chromatography (typically 99%), analyzed by liquid -paper chromatography, thin-layer chromatography, and high-performance liquid chromatography, and their lambda(max) and epsilon values are d etermined. The ability of dyes to act as affinity ligands versus Candi da boidinii FDH is evaluated by kinetic studies and determining K-D va lues from both difference spectra and enzyme inactivation studies. The parent dichlorotriazine dye VBAR binds specifically and irreversibly to FDH (k(3) 0.19 min(-1); K-D 19.3 mu M). The inactivation of the NAD (+)-dependent enzyme by VBAR is competitively inhibited by NAD(+), NAD H, and ADP. Quantitatively inhibited FDH contained approx 1 mol of dye per mole of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM each of NAD(+), NA DH, and ADP or by extensive dialysis or gel filtration chromatography, The monochlorotriazine BM dyes do not inactivate FDH but inhibit comp etitively the inactivation by VBAR, When compared to VBAR and Cibacron blue 3GA (CB3GA), all BM dye-ligands exhibited lower K-D values. FDH generally preferred binding to BM ligands which bore an aromatic termi nal biomimetic moiety substituted with a monocarboxyl group rather tha n an alpha-ketoacid. Dye binding to FDH is accompanied by a characteri stic spectral change in the range 550-800 nm. This phenomenon is pertu rbed after titration by increasing amounts of NAD(+). Electrostatic in teractions appeared to play a dominant role in the dye FDH complex. Th e BM dye-ligand bearing a m-aminobenzoate at its terminal biomimetic m oiety (BM1) exhibited the highest affinity (K-D 1.6 mu M, 8.0-fold dec rease over CB3GA). BM1 differentiated between the binding sites of FDH , displaying uncompetitive inhibition with respect to NAD(+) (K-i 15.6 mu M) and competitive with respect to formate (K-i 18.1 mu M). (C) 19 95 Academic Press, Inc.