Knuckley Bryan, Bhatia Monica, Thompson Paul R
Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, USA.
Biochemistry. 2007 Jun 5;46(22):6578-87. doi: 10.1021/bi700095s. Epub 2007 May 12.
The presumed role of an overactive protein arginine deiminase 4 (PAD4) in the pathophysiology of rheumatoid arthritis (RA) suggests that PAD4 inhibitors could be used to treat an underlying cause of RA, potentially offering a mechanism to stop further disease progression. Thus, the development of such inhibitors is of paramount importance. Toward the goal of developing such inhibitors, we initiated efforts to characterize the catalytic mechanism of PAD4 and thereby identify important mechanistic features that can be exploited for inhibitor development. Herein we report the results of mutagenesis studies as well as our efforts to characterize the initial steps of the PAD4 reaction, in particular, the protonation status of Cys645 and His471 prior to substrate binding. The results indicate that Cys645, the active site nucleophile, exists as the thiolate in the active form of the free enzyme. pH studies on PAD4 further suggest that this enzyme utilizes a reverse protonation mechanism.
蛋白精氨酸脱亚氨酶4（PAD4）活性过高在类风湿性关节炎（RA）病理生理学中的假定作用表明，PAD4抑制剂可用于治疗RA的潜在病因，这可能提供一种阻止疾病进一步发展的机制。因此，开发此类抑制剂至关重要。为了实现开发此类抑制剂的目标，我们开始努力表征PAD4的催化机制，从而确定可用于抑制剂开发的重要机制特征。在此，我们报告了诱变研究的结果以及我们对PAD4反应初始步骤进行表征的努力，特别是底物结合前Cys645和His471的质子化状态。结果表明，活性位点亲核试剂Cys645在游离酶的活性形式中以硫醇盐形式存在。对PAD4的pH研究进一步表明，该酶利用反向质子化机制。
