Department of Chemistry, University of Massachusetts, Amherst, MA, United States.
Department of Chemistry, University of Massachusetts, Amherst, MA, United States.
Methods Enzymol. 2023;679:363-380. doi: 10.1016/bs.mie.2022.08.019. Epub 2022 Sep 24.
Primary kinetic isotope effects (KIEs) provide unique insight into enzymatic reactions, as they can reveal rate-limiting steps and detailed chemical mechanisms. HIF hydroxylases, part of a family of 2-oxoglutarate (2OG) oxygenases are central to the regulation of many crucial biological processes through O-sensing, but present a challenge to monitor due to the large size of the protein substrate and the similarity between native and hydroxylated substrate. MALDI-TOF MS is a convenient tool to measure peptide masses, which can also be used to measure the discontinuous kinetics of peptide hydroxylation for Factor Inhibiting HIF (FIH). Using this technique, rate data can be observed from the mole-fraction of CTAD and CTAD-OH in small volumes, allowing noncompetitive H/D KIEs to be measured. Slow dCTAD substrate leads to extensive uncoupling of O consumption from peptide hydroxylation, leading to enzyme autohydroxylation, which is observed using UV-vis spectroscopy. Simultaneously measuring both the normal product, CTAD-OH, and the uncoupled product, autohydroxylated enzyme, the KIE on the microscopic step of hydrogen atom transfer (HAT) can be estimated. MALDI-MS analysis is a strong method for monitoring reactions that hydroxylate peptides, and can be generalized to other similar reactions, and simultaneous kinetic detection of branched products can provide valuable insight on microscopic KIEs at intermediate mechanistic steps.
初级动力学同位素效应（KIE）为酶反应提供了独特的见解，因为它们可以揭示限速步骤和详细的化学机制。HIF 羟化酶是 2-氧戊二酸（2OG）加氧酶家族的一部分，通过 O 感应在许多关键的生物过程的调节中起着核心作用，但由于蛋白质底物的体积庞大以及天然和羟化底物之间的相似性，使得对其进行监测具有挑战性。MALDI-TOF MS 是一种测量肽质量的便捷工具，也可用于测量因子抑制 HIF（FIH）的肽羟化的不连续动力学。使用该技术，可以从小体积中的 CTAD 和 CTAD-OH 的摩尔分数中观察到速率数据，从而可以测量非竞争性 H/D KIE。缓慢的 dCTAD 底物导致 O 消耗与肽羟化的广泛解偶联，导致酶自动羟化，这可以通过紫外-可见光谱观察到。同时测量正常产物 CTAD-OH 和未偶联产物自动羟化酶，可以估计氢原子转移（HAT）微观步骤上的 KIE。MALDI-MS 分析是监测肽羟化反应的有力方法，并且可以推广到其他类似的反应中，同时对分支产物的动力学检测可以提供中间机制步骤微观 KIE 的有价值的见解。
