D'Amico S, Sohier J S, Feller G
Laboratory of Biochemistry, University of Liège, Institute of Chemistry B6a, B-4000 Liège-Sart Tilman, Belgium.
J Mol Biol. 2006 May 19;358(5):1296-304. doi: 10.1016/j.jmb.2006.03.004. Epub 2006 Mar 20.
A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.
本文描述了一种新的微量量热法，该方法使用多糖和寡糖作为底物来记录α-淀粉酶的动力学参数k(cat)、K(m)和K(i)。此方法基于糖苷键水解所释放的热量。该方法已被用于研究南极嗜冷细菌产生的冷活性α-淀粉酶与其猪胰腺中最接近的结构同源物相比的活性位点特性。结果表明，嗜冷α-淀粉酶对大分子底物更具活性，并且较高的速率常数k(cat)是以对底物较低的亲和力为代价获得的。其活性位点能够容纳更大的抑制性复合物，导致麦芽糖对淀粉水解产生混合型抑制。已经开发出一种在低亲和力系统中通过等温滴定量热法记录结合焓的方法，该方法允许使用变构激活剂氯离子分析弱配体结合的能量学。结果表明，嗜冷α-淀粉酶对氯离子的低亲和力是由熵驱动的。激活剂结合的高焓和熵贡献表明冷活性酶的游离态和结合态之间存在较大的结构波动。嗜冷α-淀粉酶的动力学和热力学数据表明，参与底物结合和催化的严格保守侧链具有更高的流动性，这是其在低温下具有活性的原因，并且是由远离活性位点的稳定相互作用消失所致。
