变阻器位置,可以用各种氨基酸取代,以调整蛋白质在一系列结果中的功能,是推进个性化医学和生物工程的发展领域。目前的方法不能准确地预测哪些蛋白质含有变阻器位置或它们的取代结果。为了比较同源物中变阻器位置的患病率,我们以前在两种丙酮酸激酶(PYK)同工酶中研究了它们的发生。人肝PYK含有许多变阻器位置,可在宽范围内调节对底物磷酸烯醇丙酮酸(Kapp-PEP)的表观亲和力。相比之下,在运动发酵单胞菌PYK(ZmPYK)中未发现功能变阻器位置。Further,ZmPYK替换组包括异常大量的缺乏可测量的活性.我们假设无活性取代变体具有降低的蛋白质稳定性,排除检测到Kapp-PEP调谐。使用修改后的缓冲区,在三个位置获得了19个先前无活性的ZmPYK置换变体的稳健酶活性。令人惊讶的是,先前无活性和先前有活性的替代变体的Kapp-PEP值都接近野生型.因此,这三个位置都不是功能变阻器位置,and,与人类肝脏PYK不同,ZmPYK的Kapp-PEP通过单一替换仍然难以调节。为了直接评估对稳定性的影响,我们对所有ZmPYK置换变体进行了热变性实验。许多稳定性下降,两个增强的稳定性,这三个位置对取代的热敏感性不同,一个位置充当“稳定变阻器”。“两种PYK同源物之间的差异引发了有关潜在机制的有趣问题,这些机制允许通过某些蛋白质中的单个置换而不是其他蛋白质中的单个置换进行功能调整。
Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain
rheostat positions or their substitution outcomes. To compare the prevalence of
rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous
rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional
rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK\'s Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a \"stability rheostat.\" The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.