Narasimhulu S
Harrison Department for Surgical Research, University of Pennsylvania, Philadelphia 19104.
Endocr Res. 1991;17(1-2):209-24. doi: 10.1080/07435809109027198.
The present study offers evidence indicating that acrylamide a polar molecule inhibits substrate-binding to P450C-21 in a competitive manner and quenches tryptophanyl fluorescence in bovine adrenocortical microsomes, similar to that in the purified lipid-free enzyme. Resolution of tryptophanyl fluorescence of the microsomes revealed an acrylamide quenching constant (K2 = 9.9M, is the association constant for the quencher-fluorophore complex) which was similar to the reciprocal of its inhibition constant (1/Kj = Ka = 8.3 +- 0.9M) for substrate-binding. The substrate inhibited the fluorescence quenching by acrylamide which was in accordance with partial competition. In addition the substrate dissociation, acrylamide inhibition and fluorescence quenching constants and tryptophanyl fluorescence maximum (340-342nm) were essentially the same in the microsomes and the purified enzyme. These results suggest that, similar to that in the purified enzyme, a tryptophan in a polar environment in the membrane-bound P450, may serve as a reporter group for the substrate binding site and the site in the membrane-bound enzyme, is accessible to the substrate in aqueous phase.
本研究提供的证据表明，丙烯酰胺这种极性分子以竞争性方式抑制底物与P450C - 21的结合，并淬灭牛肾上腺皮质微粒体中的色氨酸荧光，这与纯化的无脂质酶中的情况相似。微粒体色氨酸荧光的解析显示，丙烯酰胺淬灭常数（K2 = 9.9M，K2是淬灭剂 - 荧光团复合物的缔合常数）与其底物结合抑制常数的倒数（1/Ki = Ka = 8.3±0.9M）相似。底物抑制了丙烯酰胺引起的荧光淬灭，这符合部分竞争的情况。此外，底物解离、丙烯酰胺抑制和荧光淬灭常数以及色氨酸荧光最大值（340 - 342nm）在微粒体和纯化酶中基本相同。这些结果表明，与纯化酶中的情况类似，膜结合P450中极性环境下的色氨酸可能作为底物结合位点的报告基团，并且膜结合酶中的该位点在水相中可被底物接近。
