OXALOACETATE DECARBOXYLASE - ON THE MODE OF INTERACTION WITH SUBSTRATE-MIMETIC AFFINITY LIGANDS

The mode of interaction of the ketocarboxyl-group-recognizing enzyme o xaloacetate decarboxylase (OXAD) from Pseudonomas sp., with purpose de signed (keto)carboxyl-terminal biomimetic monochlorotriazinyl-dyes (BM ) and parent dichlorotriazinyl-dye Vilmafix blue A-R (VBAR) was invest igated. Kinetic inhibition studies and determinations of K-D values of the respective dye-enzyme complex from both difference spectra and en zyme inactivation studies were employed. Substrate-mimetic (biomimetic ) dye-ligands bear a terminal (keto)carboxyl-moiety linked to the reac tive chlorotriazine ring, thus mimicking the organic acid substrate of OXAD. Dichlorotriazine-dye VBAR bound specifically and irreversibly t o OXAD (k(3) 0.22 min(-1)). The inactivation of OXAD by VBAR was enhan ced in the presence of 1 mM Mn+2 (K-D 67.2 mu M) but in the absence of metal cation was decreased (K-D 117 mu M) The metal cation behaves as a partial competitive activator. Either of binary complexes dye . OXA D and OXAD . Mn+2 could be formed first, prior to addition of the thir d constituent to form the ternary complex, although the former route m ay be favored. The pK(a) of the catalytically important nucleophile, i nvolved in the specific modification of OXAD, was calculated to 7.4. B iomimetic monochlorotriazine dyes have failed to inactivate OXAD but i nhibited competitively the inactivation by VBAR. When compared to comm ercial VBAR and Cibacron blue 3GA (CB3GA), all BM ligands show lower K -D values, therefore, higher affinity for the enzyme. OXAD preferred b inding to BM dyes which exhibited a large aliphatic ketocarboxyl-termi nal biomimetic moiety. Dye binding to OXAD was accompanied by a charac teristic spectral change in the range 550-800 nm. Electrostatic intera ctions appeared to play a dominant role in the dye . OXAD complex. The BM ligand bearing an aminoethyloxamate as its terminal biomimetic moi ety (BM7) displayed the highest affinity (K-D 0.5 or 7.0 mu M; approx 10-fold decrease over CB3GA). The BM7 ligand behaved as competitive in hibitor (K-i 98 mu M) of oxaloacetate decarboxylase against oxaloaceta te as variable substrate. (C) 1995 Academic Press, Inc.