Presnell S R, Patil G S, Mura C, Jude K M, Conley J M, Bertrand J A, Kam C M, Powers J C, Williams L D
School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA.
Biochemistry. 1998 Dec 1;37(48):17068-81. doi: 10.1021/bi981636u.
Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].
合成了新型苯甲脒芳基衍生物，并测试了它们对牛胰蛋白酶、大鼠皮肤类胰蛋白酶、人重组颗粒酶A、人凝血酶和人血浆激肽释放酶的抑制效力。所有化合物对这些蛋白酶均表现出竞争性抑制作用，其抑制常数（Ki）值在微摩尔范围内。测定了胰蛋白酶与两种对取代苯甲脒衍生物1-(4-脒基苯基)-3-(4-氯苯基)脲（ACPU）和1-(4-脒基苯基)-3-(4-苯氧基苯基)脲（APPU）形成的复合物的X射线结构，分辨率达到1.8 Å。尽管抑制剂在S1口袋之外没有直接和特异性的相互作用，但它们确实与胰蛋白酶的活性位点形成了紧密的间接接触。抑制剂通过硫酸根离子与酶相连，硫酸根离子形成了一个复杂的三中心氢键网络。将这些结构与其他具有非共价结合氧负离子的丝氨酸蛋白酶结构进行比较，发现在活性位点有一对高度保守的氧负离子结合位点。非共价结合氧负离子的位置，如硫酸根的氧原子，与四面体中间体的共价氧负离子的位置不同。非共价氧负离子的位置在“氧负离子洞”之外。动力学数据表明，质子化稳定了三元抑制剂/氧负离子/蛋白酶复合物。总之，阳离子和阴离子都可以介导抑制常数。丝氨酸蛋白酶竞争性抑制剂效力的阳离子介导作用先前已由斯特劳德及其同事报道过[卡茨，B.A.，克拉克，J.M.，芬纳-摩尔，J.S.，詹金斯，T.E.，约翰逊，C.R.，罗斯，M.J.，卢ong，C.，摩尔，W.R.，和斯特劳德，R.M.（1998年）《自然》391，608 - 612]。
