Jordan S P, Waxman L, Smith D E, Vlasuk G P
Biological Chemistry Department, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Biochemistry. 1990 Dec 18;29(50):11095-100. doi: 10.1021/bi00502a012.
Tick anticoagulant peptide (TAP) is a 60 amino acid protein which is a highly specific inhibitor of human blood coagulation factor Xa (fXa) isolated from the tick Ornithodoros moubata [Waxman, L., Smith, D. E., Arcuri, K. E., & Vlasuk, G. P. (1990) Science 248, 593-596]. Due to the limited quantities of native TAP, a recombinant version of TAP produced in Saccharomyces cerevisiae was used for a detailed kinetic analysis of the inhibition interaction with human fXa. rTAP was determined to be a reversible, slow, tight-binding inhibitor of fXa, displaying a competitive type of inhibition. The binding of rTAP to fXa is stoichiometric with a dissociation constant of (1.8 +/- 0.02) x 10(-10) M, a calculated association rate constant of (2.85 +/- 0.07) x 10(6) M-1 s-1, and a dissociation rate constant of (0.554 +/- 0.178) x 10(-3) s-1. Binding studies show that 35S-rTAP binds only to fXa and not to DFP-treated fXa or zymogen factor X, which suggests the active site of fXa is required for rTAP inhibition. That rTAP is a unique serine proteinase inhibitor is suggested both by its high specificity for its target enzyme, fXa, and also by its unique structure.
蜱抗凝肽（TAP）是一种由60个氨基酸组成的蛋白质，它是从钝缘蜱（Ornithodoros moubata）中分离出的人凝血因子Xa（fXa）的高度特异性抑制剂[Waxman, L., Smith, D. E., Arcuri, K. E., & Vlasuk, G. P. (1990) Science 248, 593 - 596]。由于天然TAP的量有限，因此使用在酿酒酵母中产生的重组TAP版本对其与人fXa的抑制相互作用进行详细的动力学分析。rTAP被确定为fXa的可逆、缓慢、紧密结合抑制剂，表现出竞争性抑制类型。rTAP与fXa的结合是化学计量的，解离常数为(1.8 +/- 0.02) x 10(-10) M，计算得到的缔合速率常数为(2.85 +/- 0.07) x 10(6) M-1 s-1，解离速率常数为(0.554 +/- 0.178) x 10(-3) s-1。结合研究表明，35S - rTAP仅与fXa结合，而不与DFP处理的fXa或酶原因子X结合，这表明fXa的活性位点是rTAP抑制所必需的。rTAP对其靶酶fXa具有高度特异性，并且其结构独特，这表明它是一种独特的丝氨酸蛋白酶抑制剂。
