Understanding how each residue position contributes to protein function has been a long‐standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions—high sequence entropy, high surface exposure, and alanine scanning—lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a “neutrality” score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

中文翻译：

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鉴定肝丙酮酸激酶中的生化中性位置。

了解每个残基位置如何影响蛋白质功能一直是蛋白质科学的长期目标。替代研究历来集中在保守的蛋白质位置上。然而，非保守位置的替换也可以改变功能。事实上，我们最近发现了在人肝丙酮酸激酶 (hLPYK) 中具有较大替代效应的非保守位置，包括改变的变构偶联。为了便于比较哪些特征决定了非保守位置何时起作用与何时不起作用，当前工作的目标是确定 hLPYK 中的中性位置。然而，现有的 hLPYK 数据表明，通常与中性位置相关的三个特征——高序列熵、高表面暴露、和丙氨酸扫描——缺乏指导实验研究所需的灵敏度。我们使用在 PYK 家族的序列比对中确定的多种进化模式来确定哪些位置的模式最少，推断这些最有可能是中性的。用总共 117 个氨基酸取代测试了 9 个位置。尽管探索所有潜在功能对于任何蛋白质都不可行，但测量了 hLPYK 变体的与底物/效应子亲和力和变构偶联相关的五个参数。对于每个位置，所有变体的总功能结果被用于量化“中性”分数。三个位置在所有五个参数上都显示出完美的中性分数。此外，9 个位置的中性分数高于变构结合位点附近的 17 个位置。因此，