Strobel S M, Szklarz G D, He Y, Foroozesh M, Alworth W L, Roberts E S, Hollenberg P F, Halpert J R
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, USA.
J Pharmacol Exp Ther. 1999 Jul;290(1):445-51.
Rabbit cytochromes P-450 (P-450) 2B4 and 2B5 differ by only 12 amino acid residues yet they exhibit unique steroid hydroxylation profiles. Previous studies have led to the identification of active site residues that are determinants of these specificities. In this study, mechanism-based inactivators were identified that discriminate between the closely related 2B4 and 2B5 enzymes. A previously characterized inhibitor, 2-ethynylnaphthalene (2EN), was found to be selective for 2B4 inactivation. As inhibitor metabolism and the partition ratio affect susceptibility, molecular dynamics simulations were performed to assess the stability of the productive binding orientation of 2EN within 2B4 and 2B5 three-dimensional models. Although 2EN was stable within the 2B4 model, it exhibited substantial movement away from the heme moiety in the 2B5 model. However, heterologously expressed 2B5 was found to catalyze the oxidation of 2EN to the stable product 2-naphthylacetic acid. Thus, the increased mobility of 2EN may result in reduced susceptibility of 2B5 by increasing the probability that the reactive ketene intermediate hydrolyzes with water instead of reacting with active site residues. Another compound, 1-adamantyl propargyl ether (1APE), selectively inactivated 2B5. The structural basis for 2EN and 1APE susceptibility was assessed using active site mutants. Interconversion of 2EN susceptibility was observed for 2B4 or 2B5 mutants containing a single alteration at residue 363. Single substitutions in 2B4 also conferred susceptibility to 1APE; however, multiple alterations were required to reduce the susceptibility of 2B5. These alterations may influence inhibitor susceptibility by affecting the stability of the productive binding orientation.
兔细胞色素P-450(P-450)2B4和2B5仅相差12个氨基酸残基,但它们表现出独特的类固醇羟基化谱。先前的研究已确定了作为这些特异性决定因素的活性位点残基。在本研究中,鉴定出了能够区分密切相关的2B4和2B5酶的基于机制的失活剂。发现一种先前已表征的抑制剂2-乙炔基萘(2EN)对2B4失活具有选择性。由于抑制剂代谢和分配比会影响敏感性,因此进行了分子动力学模拟,以评估2EN在2B4和2B5三维模型中有效结合取向的稳定性。尽管2EN在2B4模型中是稳定的,但在2B5模型中它却表现出从血红素部分大幅移动的情况。然而,发现异源表达的2B5可催化2EN氧化为稳定产物2-萘乙酸。因此,2EN流动性的增加可能通过增加反应性乙烯酮中间体与水水解而不是与活性位点残基反应的概率,导致2B5的敏感性降低。另一种化合物1-金刚烷基炔丙基醚(1APE)选择性地使2B5失活。使用活性位点突变体评估了2EN和1APE敏感性的结构基础。在残基363处含有单个改变的2B4或2B5突变体中观察到2EN敏感性的相互转换。2B4中的单个取代也赋予了对1APE的敏感性;然而,需要多个改变才能降低2B5的敏感性。这些改变可能通过影响有效结合取向的稳定性来影响抑制剂的敏感性。