Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States.
Texas A&M University-Kingsville, Kingsville, Texas 78363, United States.
Biochemistry. 2021 Dec 21;60(50):3822-3828. doi: 10.1021/acs.biochem.1c00558. Epub 2021 Dec 7.
Evolution of dihydrofolate reductase (DHFR) has been studied using the enzyme from DHFR (ecDHFR) as a model, but less studies have used the enzyme from DHFR (hsDHFR). Each enzyme maintains a short and narrow distribution of hydride donor-acceptor distances (DAD) at the tunneling ready state (TRS). Evolution of the enzyme was previously studied in ecDHFR where three key sites were identified as important to the catalyzed reaction. The corresponding sites in hsDHFR are F28, 62-PEKN, and 26-PPLR. Each of these sites was studied here through the creation of mutant variants of the enzyme and measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs) on the reaction. F28 is mutated first to M (F28M) and then to the L of the bacterial enzyme (F28L). The KIEs of the F28M variant are larger and more temperature-dependent than wild-type (WT), suggesting a broader and longer average DAD at the TRS. To more fully mimic ecDHFR, we also study a triple mutant of the human enzyme (F32L-PP26N-PEKN62G). Remarkably, the intrinsic KIEs, while larger in magnitude, are temperature-independent like the WT enzymes. We also construct deletion mutations of hsDHFR removing both the 62-PEKN and 26-PPLR sequences. The results mirror those described previously for insertion mutants of ecDHFR. Taken together, these results suggest a balancing act during DHFR evolution between achieving an optimal TRS for hydride transfer and preventing product inhibition arising from the different intercellular pools of NADPH and NADP in prokaryotic and eukaryotic cells.
二氢叶酸还原酶（DHFR）的进化已使用来自 DHFR（ecDHFR）的酶作为模型进行了研究，但使用来自 DHFR（hsDHFR）的酶的研究较少。每种酶在隧穿准备状态（TRS）下保持短而窄的氢化物供体-受体距离（DAD）分布。以前在 ecDHFR 中研究了酶的进化，其中确定了三个关键位点对催化反应很重要。hsDHFR 中的对应位点是 F28、62-PEKN 和 26-PPLR。在这里，通过创建酶的突变变体并测量反应的固有动力学同位素效应（KIE）对温度的依赖性来研究这些位点中的每一个。首先将 F28 突变为 M（F28M），然后突变为细菌酶的 L（F28L）。F28M 变体的 KIE 比野生型（WT）更大且对温度更敏感，表明 TRS 处的平均 DAD 更宽且更长。为了更完全地模拟 ecDHFR，我们还研究了人类酶的三重突变体（F32L-PP26N-PEKN62G）。值得注意的是，固有 KIE 虽然幅度更大，但与 WT 酶一样对温度不敏感。我们还构建了去除 62-PEKN 和 26-PPLR 序列的 hsDHFR 缺失突变。结果与之前描述的 ecDHFR 插入突变相似。总而言之，这些结果表明在 DHFR 进化过程中，在为氢化物转移实现最佳 TRS 和防止来自原核细胞和真核细胞中不同细胞间 NADPH 和 NADP 池的产物抑制之间存在平衡行为。
