| heal.abstract |
The mode of interaction of two oxalate-recognizing enzymes, oxalate oxidase (OXO) and oxalate decarboxylase (OXD), with carboxyl-terminal biomimetic monochlorotriazinyl dyes (BM) was studied. Determinations of K(D) values of the respective dye-enzyme complexes by difference spectra, and kinetic inhibition studies, were employed. Oxalate-mimetic (biomimetic) dye-ligands bear a terminal carboxyl-moiety linked to the reactive chlorotriazine ring, thus mimicking the organic acid substrate of the enzymes. OXO preferred binding to BM dyes which exhibited carboxyl-aromatic terminal functions, whereas OXD has shown preference for dye-ligands bearing terminal aliphatic biomimetic moieties of moderate length. Dye binding to OXO and OXD was accompanied by a characteristic spectral change in the range 500-850 nm. Mixed-type forces (electrostatic and hydrophobic) are present in the dye OXO complex, whereas electrostatic interactions play a dominant role in the dye OXD complex. Biomimetic dyes bearing a m-aminobenzoate (BM1) and mercaptopyruvate (BM6) at the terminal biomimetic moiety, exhibited the highest affinity for oxalate oxidase and oxalate decarboxylase, respectively. These dyes, when compared with commercial Cibacron blue 3GA, show a decrease of their K(D) with OXO and OXD by 22- and 35-fold, respectively. The BM1 ligand behaved as non-linear mixed type inhibitor of OXO with respect to oxalate (K(i) 5.1 μM for the OXO BM1 complex, and K(i) 0.2 μM for the BM1 OXO oxalate complex), whereas BM6 behaved as competitive inhibitor of OXD against oxalate as variable substrate (K(i) 25.4 μM). |
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