Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43124, Parma, Italy.
Department of Medicine and Surgery, University of Parma, 43126, Parma, Italy.
Anal Biochem. 2020 Mar 15;593:113595. doi: 10.1016/j.ab.2020.113595. Epub 2020 Jan 24.
Steady-state enzyme kinetics typically relies on the measurement of 'initial rates', obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or 'lag phase'. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s.
稳态酶动力学通常依赖于“初始速率”的测量，即在底物未被显著消耗且产物形成量可忽略不计的情况下获得。虽然初始速率通常比反应时间进程中稍后测量的速率快，但有时反应的速度似乎反而会随时间增加，在初始延迟或“滞后期”之后才达到稳定水平。这种行为需要由实验人员进行解释。为了协助解释，本文分析了在酶测定中，观察到的速率在开始时较慢，然后逐渐加速的许多原因。可能的原因从琐碎的伪影到更深层次的机制或生物物理因素的情况都有涉及。我们提供了大多数这些原因的实际示例，首先基于在我们实验室中研究的鸟氨酸 δ-氨基转移酶和其他依赖吡哆醛-5-磷酸的酶进行的实验。在这个调查的旁边，我们提供了证据表明，鸟氨酸 δ-氨基转移酶反应的产物谷氨酸 5-半醛醛自发环化生成吡咯啉 5-羧酸，其速率常数大于 3 s。
