Oxaloacetate decarboxylase: on the mode of interaction with substrate-mimetic affinity ligands.
The mode of interaction of the ketocarboxyl-group-recognizing enzyme oxaloacetate decarboxylase (OXAD) from Pseudonomas sp., with purpose-designed (keto)-carboxyl-terminal biomimetic monochlorotriazinyl-dyes (BM) and parent dichlorotriazinyl-dye Vilmafix blue A-R (VBAR) was investigated. Kinetic inhibition studies and determinations of KD values of the respective dye-enzyme complex from both difference spectra and enzyme inactivation studies were employed. Substratemimetic (biomimetic) dye-ligands bear a terminal (keto)carboxyl-moiety linked to the reactive chlorotriazine ring, thus mimicking the organic acid substrate of OXAD. Dichlorotriazine-dye VBAR bound specifically and irreversibly to OXAD (k3 0.22 min-1). The inactivation of OXAD by VBAR was enhanced in the presence of 1 mM Mn+2 (KD 67.2 microM) but in the absence of metal cation was decreased (KD 117 microM). The metal cation behaves as a partial competitive activator. Either of binary complexes dye.OXAD and OXAD.Mn+2 could be formed first, prior to addition of the third constituent to form the ternary complex, although the former route may be favored. The pKa of the catalytically important nucleophile, involved in the specific modification of OXAD, was calculated to 7.4. Biomimetic monochlorotriazine dyes have failed to inactivate OXAD but inhibited competitively the inactivation by VBAR. When compared to commercial VBAR and Cibacron blue 3GA (CB3GA), all BM ligands show lower KD values, therefore, higher affinity for the enzyme. OXAD preferred binding to BM dyes which exhibited a large aliphatic ketocarboxyl-terminal biomimetic moiety. Dye binding to OXAD was accompanied by a characteristic spectral change in the range 550-800 nm. Electrostatic interactions appeared to play a dominant role in the dye.OXAD complex. The BM ligand bearing an aminoethyloxamate as its terminal biomimetic moiety (BM7) displayed the highest affinity (KD 0.5 or 7.0 microM; approx 10-fold decrease over CB3GA). The BM7 ligand behaved as competitive inhibitor (Ki 98 microM) of oxaloacetate decarboxylase against oxaloacetate as variable substrate.
Labrou
N E
NE
Department of Agricultural Biology and Biotechnology, Agricultural University of Athens, Greece.
Clonis
Y D
YD
D000880:Anthraquinones / Q000494:pharmacology*; D001667:Binding, Competitive; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D004396:Coloring Agents / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D008345:Manganese / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014227:Triazines / Q000494:pharmacology*
D000880:Anthraquinones; D002413:Cations, Divalent; D004396:Coloring Agents; D008024:Ligands; D014227:Triazines; C091762:Vilmafix Blue A-R; D008345:Manganese; C012842:Cibacron Blue F 3GA; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase