Fisher H F, Maniscalco S, Singh N, Adediran S A
Department of Biochemistry, University of Kansas School of Medicine, Kansas City 64128.
J Biol Chem. 1988 Aug 25;263(24):11704-10.
The usefulness of the indicator dye method for the detection of transient enzyme-product intermediates has been very limited due to the near impossibility of resolving the apparent single exponential time courses resulting from sequences of steps linked by rather similar rate constants. We propose here a novel approach, the proton-product time course method, a procedure which can extract a great deal of the mechanistic information which remains buried in conventional proton release-time course measurements. The method involves nothing more than measuring the ratio, r, of the moles of H+ released to the moles of product formed as a function of time. We derive the theory relating this r function to mechanisms of varying complexity, explore the theoretical behavior of the function in various possible mechanistic situations, and employ the new approach in an experimental system. We demonstrate the fact that the proton/product time course ratio method can provide evidence of the existence of hidden steps in transient state kinetic studies, that it can determine accurate thermodynamic pK values of the intermediate complexes involved in those steps, and that it can produce time courses of individual intermediates which are obscure to conventional kinetic methods.
由于难以分辨由速率常数颇为相似的一系列步骤所产生的看似单一的指数时间进程，指示染料法用于检测瞬态酶 - 产物中间体的实用性非常有限。我们在此提出一种新方法，即质子 - 产物时间进程法，该方法能够从传统质子释放时间进程测量中难以挖掘的大量机制信息中提取出来。该方法仅仅是测量作为时间函数的H⁺释放摩尔数与产物形成摩尔数的比率r。我们推导了将此r函数与不同复杂程度机制相关联的理论，探讨了该函数在各种可能机制情况下的理论行为，并在一个实验系统中应用了这种新方法。我们证明了质子/产物时间进程比率法能够在瞬态动力学研究中为隐藏步骤的存在提供证据，能够确定这些步骤中涉及的中间复合物的准确热力学pK值，并且能够生成传统动力学方法难以察觉的各个中间体的时间进程。
