BIOMIMETIC DYE-LIGANDS FOR OXALATE-RECOGNIZING ENZYMES - STUDIES WITHOXALATE OXIDASE AND OXALATE DECARBOXYLASE

The mode of interaction of two oxalate-recognizing enzymes, oxalate ox idase (OXO) and oxalate decarboxylase (OXD), with carboxyl-terminal bi omimetic monochlorotriazinyl dyes (BM) was studied. Determinations of K-D values of the respective dye-enzyme complexes by difference spectr a, and kinetic inhibition studies, were employed. Oxalate-mimetic (bio mimetic) dye-ligands bear a terminal carboxyl-moiety linked to the rea ctive chlorotriazine ring, thus mimicking the organic acid substrate o f the enzymes. OXO preferred binding to BM dyes which exhibited carbox yl-aromatic terminal functions, whereas OXD has shown preference for d ye-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 (electrost atic and hydrophobic) are present in the dye . OXO complex, whereas el ectrostatic 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 affini ty 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 B M1 ligand behaved as non-linear mixed type inhibitor of OXO with respe ct to oxalate (K-i 5.1 mu M for the OXO . BM1 complex, and K-i' 0.2 mu M for the BM1 . OXO . oxalate complex), whereas BM6 behaved as compet itive inhibitor of OXD against oxalate as variable substrate (K-i 25.4 mu M).