Abstract：
BAHD acyl-coenzyme A (CoA) acyltransferases play key roles in a large number of biosynthetic reactions involved in plant specialized metabolism. One approach to measure reaction rates for these enzymes is to quantify the amide or ester reaction products following chromatographic separation of reaction components, an approach that can be labor intensive and time consuming, and complicated by a lack of pure standards. We previously developed and validated an alternative approach using 5,5\'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman\'s reagent) to spectrophotometrically monitor reaction progress by the release of free CoA in the reaction. This approach allows near-real time measurement of reaction rates, permitting reaction conditions (buffer, reactant, and enzyme concentrations, etc.) to be changed \"on the fly.\" The ease and rapidity of data collection allows a high density of data points to be collected for determination of kinetic parameters. Here we provide a detailed procedure for using DTNB to measure BAHD acyl-CoA acyltransferase reaction rates, and as an example, use it to determine kinetic parameters for red clover hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase, a BAHD acyl-CoA hydroxycinnamoyltransferase not previously characterized with respect to kinetic parameters. This approach may be more generally applicable to transferases using CoA donors.
摘要：
BAHD酰基辅酶A（CoA）酰基转移酶在涉及植物专门代谢的大量生物合成反应中起关键作用。测量这些酶的反应速率的一种方法是在反应组分的色谱分离后量化酰胺或酯反应产物。这种方法可能是劳动密集型和耗时的，由于缺乏纯粹的标准而变得复杂。我们先前使用5,5'-二硫代-双-（2-硝基苯甲酸）（DTNB，Ellman试剂）通过反应中游离CoA的释放来分光光度法监测反应进程。这种方法允许近实时测量反应速率，允许反应条件(缓冲液，反应物,和酶浓度，等。)要在飞行中改变\“。“数据收集的方便性和快速性允许收集高密度的数据点以确定动力学参数。在这里，我们提供了使用DTNB测量BAHD酰基辅酶A酰基转移酶反应速率的详细程序，作为一个例子，用它来确定红三叶草羟基肉桂酰辅酶A：莽草酸羟基肉桂酰转移酶的动力学参数，BAHD酰基-CoA羟基肉桂酰转移酶先前未就动力学参数进行表征。这种方法可以更普遍地适用于使用CoA供体的转移酶。
