Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, USA.
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA.
J Biol Chem. 2021 Jan-Jun;296:100059. doi: 10.1074/jbc.RA120.014057. Epub 2020 Nov 24.
Inositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases.
肌醇多磷酸 1-磷酸酶（INPP1）是金属依赖/锂抑制磷酸单酯酶蛋白家族的原型成员，该家族由保守的三维核心结构定义。该家族中的酶在不同的途径中发挥作用，包括肌醇信号转导、糖异生和硫同化。我们使用结构和生化研究报告了底物和锂对 INPP1 催化中心内金属结合位点网络的影响。我们发现，只有在添加底物或产物时，锂才会优先占据参与金属活化的关键位点。与野生型相比，突变一个保守残基，该残基选择性地协调假定的锂结合位点，会导致抑制常数降低 100 倍。此外，我们报告了 INPP1/肌醇 1,4-二磷酸复合物，该复合物阐明了酶活性位点的关键特征。我们的结果为非竞争抑制和底物识别提供了结构基础，并定义了该调节磷酸酶家族中金属结合的序列基序。
