Proton Inventory for Reactions in Blood Clotting

描述（由申请人提供）：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最复杂的成员。对三种底物的酶催化水解进行全溶剂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残基的效果。2)为了测试P1-P5和大约‘-P5’位点的集体贡献，但没有外源位点，将用凝血酶研究含有n端2-氨基苯甲酰(AB)-Val荧光团和c端lys - 2,4 -二硝基苯（DNP）猝灭剂和aspoh以增强溶解度的荧光猝灭底物。3) n -酰基或离去基结合位点对质子参与程度的外源依赖性将通过选定的自然反应的研究来评估。天然存在的凝血酶底物在远离活性位点的指定位点与酶特异性结合。首先进行初步实验，找出测量Michaelis Menten参数的最佳条件，然后在H2O和D20条件下研究Michaelis Menten参数对pH的依赖关系。在8到10种同位素水的混合物中得到的特定动力学参数（kcat和kcat /Km），每种混合物在n，介质中D的特定原子分数，将适合于从Gross-Butlet方程导出的模型。将计算过渡态多质子转移和溶剂贡献的分馏因子。统计上最显著的模型将使用？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。

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.