Proton Inventory for Reactions in Blood Clotting

凝血反应的质子清单

• 批准号: 6503163
• 负责人: ILDIKO M KOVACH
• 金额: $ 15.31万
• 依托单位: CATHOLIC UNIVERSITY OF AMERICA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6503163
• 关键词: active sites; binding sites; blood coagulation; clotting factor; coagulation factor X; endopeptidases; enzyme activity; enzyme substrate; high performance liquid chromatography; hydrogen ions; hydrolysis; plasmin; protein binding; thrombin

DESCRIPTION (provided by applicant): The main goal of this proposal is to characterize the number of protonic sites and their mode of participation in the catalysis of substrate hydrolysis by hemostatic and thrombolytic enzymes. The target enzymes are human thrombin, human factor Xa, activated protein C (APC) and bovine plasmin, some of the most sophisticated members of the serine protease family. Characterization of full and partial solvent isotope effects will be carried out for the enzyme-catalyzed hydrolysis of sets of three types of substrates: 1) Chromogenic or fluorogenic di- to pentapeptide amide substrates will test the effect of P1-P5 residues. 2) To test the collective contribution of P1-P5 and about '-P5' sites, but without exosites, fluorescence-quenched substrates with an N-terminal 2-aminobenzoyl (AB)-Val fluorophore and a C-terminal Lys-2, 4-dinitrophenyl (DNP) quencher and an Asp-OH to enhance solubility, will be studied with thrombin. 3) The exosite dependence of the effect of the N-acyl, or leaving group binding site on the extent of protonic participation will be evaluated from studies of selected natural reactions. Naturally occurring substrates of thrombin achieve specific binding to the enzyme at designated exosites remote from the active site. Preliminary experiments will be conducted to find the optimal conditions for the measurements of Michaelis Menten parameters and then their dependence on pH will be studied in H2O and D20. Specific kinetic parameters (kcat and kcat /Km) obtained in 8 to 10 mixtures of isotopic waters each at n, a particular atom fraction of D in the medium, will be fit to models derived from the Gross-Butlet equation. Fractionation factors for multiproton transfer at the transition state (s) and for solvent contribution will be calculated. The statistically most significant model will be sought using the ?2 and F-tests. The results of these studies will promote the basic understanding of enzyme catalytic power and enzyme evolutionary theory, and also provide guidelines to the development of transition state analog inhibitors in targeted drug design.

描述（由申请方提供）：本提案的主要目标是表征质子位点的数量及其参与止血酶和溶栓酶催化底物水解的模式。靶酶是人凝血酶、人Xa因子、活化蛋白C（APC）和牛纤溶酶，它们是丝氨酸蛋白酶家族中一些最复杂的成员。将对三种类型底物的酶催化水解进行完全和部分溶剂同位素效应的表征：1）显色或荧光二肽至五肽酰胺底物将测试P1-P5残基的效应。2)为了测试P1-P5和大约“-P5”位点的集体贡献，但没有外部位点，将用凝血酶研究具有N-末端2-氨基苯甲酰基（AB）-瓦尔荧光团和C-末端Lys-2，4-二硝基苯基（DNP）猝灭剂和Asp-OH以增强溶解度的荧光猝灭底物。3)将从选定的自然反应的研究中评估N-酰基或离去基团结合位点对质子参与程度的影响的外部位点依赖性。天然存在的凝血酶底物在远离活性位点的指定外部位点处实现与酶的特异性结合。将进行初步实验以找到测量Michaelis Menten参数的最佳条件，然后在H2O和D20中研究它们对pH的依赖性。具体的动力学参数（kcat和kcat /Km），在8至10个混合物的同位素沃茨，每个在n，一个特定的原子分数的D在介质中，将适合的模型来自格罗斯-巴特勒方程。将计算过渡态多质子转移和溶剂贡献的分馏因子。将使用？2和F检验。这些研究结果将促进对酶催化能力和酶进化理论的基本认识，并为靶向药物设计中过渡态类似物抑制剂的开发提供指导。