Proton Inventory for Reactions in Blood Clotting

描述(由申请人提供)：本提案的主要目的是表征质子中心的数量及其参与止血和溶栓酶催化底物水解的方式。目标酶是人凝血酶、人凝血因子Xa、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。将对酶催化的三种底物组的全部和部分溶剂同位素效应进行表征：1)发色或荧光二至五肽酰胺底物将检验P1-P5残基的效果。2)为了测试P1-P5和大约‘-P5’位的集体贡献，但没有外显子，将用凝血酶研究带有N-端2-氨基苯甲酰基(AB)-Val荧光团和C-端Lys-2，4-二硝基苯基(DNP)猝灭剂和Asp-OH以提高溶解性的荧光猝灭底物。3)N-酰基或离开基团结合位置对质子参与程度的影响的外在性依赖将通过对选定的自然反应的研究来评估。天然产生的凝血酶底物在远离活性部位的指定外切点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的依赖关系。在8-10种同位素水混合物中获得的特定动力学参数(kcat和kcat/Km)将符合从Gross-Butlet方程导出的模型。将计算多质子在过渡态转移(S)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检验来寻找。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.