Wells C M, Di Cera E
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1992 Dec 1;31(47):11721-30. doi: 10.1021/bi00162a008.
The amidase activity of human alpha-thrombin has been studied at steady state as a function of the concentration of several chloride salts, at a constant ionic strength I = 0.2 M. All kinetic steps of the catalytic mechanism of the enzyme have been solved by studies conducted as a function of relative viscosity of the solution. Among all monovalent cations, Na+ is the most effective in activating thrombin catalysis. This effect is observed with different amide substrates and also with gamma-thrombin, a proteolytic derivative of the native enzyme which has little clotting activity but retains amidase activity toward small synthetic substrates. The specific effects observed as a function of Na+ concentration are indicative of a binding interaction of this monovalent cation with the enzyme. The basis of this interaction has been explored by measurements of substrate hydrolysis collected in a three-dimensional matrix of substrate concentration, relative viscosity, and Na+ concentration, keeping the ionic strength constant with an inert cation such as choline or tetraethylammonium. The data have globally been analyzed in terms of a kinetic linkage scheme where Na+ plays the role of an allosteric effector. The properties of the enzyme change drastically upon binding of Na+, with substrate binding and dissociation, as well as deacylation, occurring on a time scale which is 1 order of magnitude faster. The apparent association constants for Na+ binding to the various intermediate forms of the enzyme have all been resolved from analysis of experimental data and are in the range of 50-100 M-1 at 25 degrees C. Studies conducted at different temperatures, in the range 15-35 degrees C, have revealed the enthalpic and entropic components of Na+ binding to the enzyme. The results obtained from steady-state measurements are supported by independent measurements of the intrinsic fluorescence of the enzyme as a function of Na+ concentration at a constant ionic strength I = 0.2 M, over the temperature range 15-35 degrees C. These measurements are indicative of a drastic conformational change of the enzyme upon Na+ binding to a single site. The energetics of Na+ binding derived from analysis of fluorescence measurements agree very well with those derived independently from steady-state determinations. It is proposed that thrombin exists in two conformations, slow and fast, and that the slow-->fast transition is triggered by binding of a monovalent cation. The high specificity in thrombin activation found in the case of Na+ is the result of its higher affinity compared to all other monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)
在恒定离子强度I = 0.2 M的条件下，研究了人α-凝血酶的酰胺酶活性与几种氯化物盐浓度的关系。通过研究酶催化机制的所有动力学步骤与溶液相对粘度的函数关系，解决了这些问题。在所有单价阳离子中，Na+对激活凝血酶催化作用最为有效。在不同的酰胺底物以及γ-凝血酶（天然酶的一种蛋白水解衍生物，几乎没有凝血活性，但对小的合成底物仍保留酰胺酶活性）中均观察到了这种效应。观察到的与Na+浓度相关的特定效应表明该单价阳离子与酶存在结合相互作用。通过在底物浓度、相对粘度和Na+浓度的三维矩阵中收集底物水解的测量数据，探索了这种相互作用的基础，同时用胆碱或四乙铵等惰性阳离子保持离子强度恒定。根据动力学联系方案对数据进行了整体分析，其中Na+起着变构效应剂的作用。Na+结合后，酶的性质发生了巨大变化，底物的结合和解离以及脱酰作用在时间尺度上加快了1个数量级。通过对实验数据的分析，确定了Na+与酶的各种中间形式结合的表观缔合常数，在25℃时其范围为50 - 100 M-1。在15 - 35℃范围内不同温度下进行的研究揭示了Na+与酶结合的焓和熵成分。在恒定离子强度I = 0.2 M下，在15 - 35℃温度范围内，通过独立测量酶的固有荧光与Na+浓度的函数关系，支持了稳态测量得到的结果。这些测量表明，Na+与单个位点结合后，酶发生了剧烈的构象变化。荧光测量分析得出的Na+结合能与稳态测定独立得出的结果非常吻合。有人提出，凝血酶存在两种构象，即慢型和快型，单价阳离子的结合触发了慢型向快型的转变。在Na+的情况下，凝血酶激活具有高特异性，这是因为它与所有其他单价阳离子相比具有更高的亲和力。（摘要截断于４００字）
