Cho Uhn Soo, Park Eun Young, Dong Mi Sook, Park Bum Seok, Kim Keehyuk, Kim Kyung Hyun
Graduate School of Biotechnology, Korea University, 5-1 Anam dong, Sungbuk-gu, Seoul 136-701, South Korea.
Biochim Biophys Acta. 2003 May 30;1648(1-2):195-202. doi: 10.1016/s1570-9639(03)00148-1.
Human cytochrome P450 (P450) enzymes exhibit remarkable diversity in their substrate specificities, participating in oxidation reactions of a wide range of xenobiotic drugs. Previously, we reported that alpha-naphthoflavone (ANF) is bound to the recombinant P450 1A2 tightly and stabilizes an overall enzyme conformation. The present study is designed to determine the type of P450 1A2 inhibition exerted by ANF, using two different substrates of P450 1A2, 7-ethoxycoumarin (EOC) and 7-ethoxyresorufin (EOR). ANF is generally known as a competitive inhibitor of the enzyme. However, in our tight-binding enzyme kinetics study, ANF acts as noncompetitive inhibitor in 7-ethoxycoumarin O-deethylation (ECOD) (K(i)=55.0 nM), but as competitive inhibitor in 7-ethoxyresorufin O-deethylation (EROD) (K(i)=1.4 nM). Based on homology modeling studies, ANF is positioned to bind to a hydrophobic cavity next to the active site where it may cause a direct effect on substrate binding. It is agreed with the predicted binding site of ANF in P450 3A4, in which ANF is rather known as a stimulating modulator. Our results suggest that ANF binds near the active site of P450 1A2 and exhibits differential inhibition mechanisms, possibly depending on the molecular structure of the substrate.
人类细胞色素P450（P450）酶在底物特异性方面表现出显著的多样性，参与多种外源性药物的氧化反应。此前，我们报道了α-萘黄酮（ANF）与重组P450 1A2紧密结合，并稳定了整个酶的构象。本研究旨在使用P450 1A2的两种不同底物7-乙氧基香豆素（EOC）和7-乙氧基试卤灵（EOR）来确定ANF对P450 1A2的抑制类型。ANF通常被认为是该酶的竞争性抑制剂。然而，在我们的紧密结合酶动力学研究中，ANF在7-乙氧基香豆素O-脱乙基化（ECOD）反应中表现为非竞争性抑制剂（K(i)=55.0 nM），但在7-乙氧基试卤灵O-脱乙基化（EROD）反应中表现为竞争性抑制剂（K(i)=1.4 nM）。基于同源建模研究，ANF位于活性位点旁边的疏水腔中，在那里它可能对底物结合产生直接影响。这与ANF在P450 3A4中的预测结合位点一致，在P450 3A4中，ANF更被认为是一种刺激调节剂。我们的结果表明，ANF结合在P450 1A2活性位点附近，并表现出不同的抑制机制，这可能取决于底物的分子结构。
