Anophelin: Kinetics and Mechanism of Thrombin Inhibition

凝血酶 化学 离解常数 水蛭素 立体化学 结合位点 动力学 非竞争性抑制 生物物理学 生物化学 血小板 受体 生物 免疫学 物理 量子力学
作者
Ivo M.B. Francischetti,Jesús G. Valenzuela,José M. C. Ribeiro
出处
期刊:Biochemistry [American Chemical Society]
卷期号:38 (50): 16678-16685 被引量:104
标识
DOI:10.1021/bi991231p
摘要

Anophelin is a 6.5-kDa peptide isolated from the salivary gland of Anopheles albimanus that behaves as an alpha-thrombin inhibitor. In this paper, kinetic analyses and the study of mechanism of alpha-thrombin inhibition by anophelin were performed. Anophelin was determined to be a reversible, slow, tight-binding inhibitor of alpha-thrombin, displaying a competitive type of inhibition. The binding of anophelin to alpha-thrombin is stoichiometric with a dissociation constant (K(i)) of 5.87 +/- 1.46 pM, a calculated association rate constant (k(1)) of 2.11 +/- 0.06 x 10(8) M(-1) s(-1), and a dissociation rate constant (k(-1)) of 4.05 +/- 0.97 x 10(-4) s(-1). In the presence of 0.15 and 0.4 M NaCl, a 17.6- and 207-fold increase in the K(i) of anophelin-alpha-thrombin complex was observed, respectively, indicating that ionic interactions are important in anophelin-alpha-thrombin complex formation. Incubation of alpha-thrombin with C-terminal hirudin fragment 54-65 that binds to alpha-thrombin anion binding exosite 1 (TABE1) attenuates alpha-thrombin inhibition by anophelin; anophelin also blocks TABE1-dependent trypsin-mediated proteolysis of alpha-thrombin. Using gamma-thrombin, an alpha-thrombin derivative where the anion binding exosite has been disrupted, anophelin behaves as a fast and classical competitive inhibitor of gamma-thrombin hydrolysis of small chromogenic substrate (K(i) = 0. 694 +/- 0.063 nM). In addition, anophelin-gamma-thrombin complex formation is prevented by treatment of the enzyme with D-Phe-Pro-Arg-chloromethyl ketone (PPACK), a reagent that irreversibly blocks the catalytic site of thrombin. It is concluded that anophelin is a potent dual inhibitor of alpha-thrombin because it binds both to TABE1 and to the catalytic site, optimal binding being dependent on the availability of both domains. Finally, anophelin inhibits clot-bound alpha-thrombin with an IC(50) of 45 nM and increases the lag phase that precedes explosive in vitro alpha-thrombin generation after activation of intrinsic pathway of blood coagulation. Because of its unique primary sequence, anophelin may be used as a novel reagent to study the structure and function of alpha-thrombin.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
Doctor_mao完成签到,获得积分10
5秒前
量子星尘发布了新的文献求助10
5秒前
6秒前
AI_S应助高序采纳,获得20
8秒前
野山发布了新的文献求助10
10秒前
11秒前
晓畅完成签到,获得积分10
12秒前
Unbelievable完成签到,获得积分10
13秒前
越瑟淳潔完成签到,获得积分10
14秒前
15秒前
静默向上发布了新的文献求助10
17秒前
17秒前
独特冰安发布了新的文献求助10
17秒前
SciGPT应助ZM采纳,获得10
17秒前
阿瞒完成签到 ,获得积分10
18秒前
Jaaay发布了新的文献求助10
19秒前
20秒前
21秒前
21秒前
SamYang发布了新的文献求助10
22秒前
li发布了新的文献求助10
24秒前
钫人完成签到,获得积分10
24秒前
风趣的胜应助Jaaay采纳,获得10
25秒前
27秒前
wjx关闭了wjx文献求助
27秒前
Simlove发布了新的文献求助10
28秒前
Orange应助SamYang采纳,获得10
29秒前
30秒前
AJJACKY完成签到,获得积分10
30秒前
31秒前
wjx关闭了wjx文献求助
32秒前
11应助DAWOUD采纳,获得10
32秒前
chl完成签到,获得积分20
33秒前
33秒前
zsy发布了新的文献求助30
35秒前
wjx关闭了wjx文献求助
36秒前
37秒前
爱吃辣椒的蓉蓉完成签到 ,获得积分10
37秒前
37秒前
陈晶完成签到 ,获得积分10
39秒前
高分求助中
A new approach to the extrapolation of accelerated life test data 1000
Picture Books with Same-sex Parented Families: Unintentional Censorship 700
ACSM’s Guidelines for Exercise Testing and Prescription, 12th edition 500
Nucleophilic substitution in azasydnone-modified dinitroanisoles 500
不知道标题是什么 500
Indomethacinのヒトにおける経皮吸収 400
Phylogenetic study of the order Polydesmida (Myriapoda: Diplopoda) 370
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 3975378
求助须知:如何正确求助?哪些是违规求助? 3519775
关于积分的说明 11199621
捐赠科研通 3256067
什么是DOI,文献DOI怎么找? 1798124
邀请新用户注册赠送积分活动 877386
科研通“疑难数据库(出版商)”最低求助积分说明 806305