Labrou N E, Clonis Y D
Department of Agricultural Biology and Biotechnology, Agricultural University of Athens, Greece.
Arch Biochem Biophys. 1995 Aug 1;321(1):61-70. doi: 10.1006/abbi.1995.1368.
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.
研究了来自假单胞菌属的酮羧基识别酶草酰乙酸脱羧酶（OXAD）与专门设计的（酮）羧基末端仿生单氯三嗪基染料（BM）和母体二氯三嗪基染料维尔马菲克斯蓝A-R（VBAR）的相互作用模式。采用动力学抑制研究以及通过差异光谱和酶失活研究来测定各自染料-酶复合物的KD值。底物模拟（仿生）染料配体带有与反应性氯三嗪环相连的末端（酮）羧基部分，从而模拟OXAD的有机酸底物。二氯三嗪染料VBAR与OXAD特异性且不可逆地结合（k3为0.22 min-1）。在1 mM Mn+2存在下，VBAR对OXAD的失活作用增强（KD为67.2 microM），但在没有金属阳离子时失活作用减弱（KD为117 microM）。金属阳离子表现为部分竞争性激活剂。在添加第三种成分形成三元复合物之前，可以先形成二元复合物染料.OXAD和OXAD.Mn+2中的任何一种，尽管前一种途径可能更受青睐。参与OXAD特异性修饰的催化重要亲核试剂的pKa经计算为7.4。仿生单氯三嗪染料未能使OXAD失活，但竞争性抑制了VBAR导致的失活。与市售的VBAR和汽巴克隆蓝3GA（CB3GA）相比，所有BM配体的KD值都更低，因此对该酶具有更高的亲和力。OXAD更倾向于与具有大的脂肪族酮羧基末端仿生部分的BM染料结合。染料与OXAD的结合伴随着550 - 800 nm范围内的特征光谱变化。静电相互作用似乎在染料.OXAD复合物中起主导作用。带有氨基乙基草氨酸作为其末端仿生部分的BM配体（BM7）表现出最高的亲和力（KD为0.5或7.0 microM；比CB3GA降低约10倍）。BM7配体作为草酰乙酸脱羧酶以草酰乙酸作为可变底物的竞争性抑制剂（Ki为98 microM）。
