Design of molecular switches of glutamate dehydrogenase based on intramolecular electric field regulation

Abstract This study focuses on designing molecular switches to regulate the activity of glutamate dehydrogenase from Thermus thermophilus (3AOG) by altering its intramolecular electric field. By molecular dynamics simulations and interaction network analyses, critical residues involved in the proton‐coupled electron transfer pathway were identified, revealing their roles in interdomain and interfacial electron transfer within the enzyme. Key residues with significant remote electrostatic effects on the electric field at the proton donor (K111) and the catalytic site (D151) were defined. Residues contributing more than 1 MV cm −1 to the electric field at the active site were identified as primary influencers of enzyme activity. Two molecular switches were designed: positive switch (3AOG‐6NZX‐PM) and negative switch (3AOG‐6NZX‐NM). Experimental validation showed that the positive molecular switch enhanced enzymatic activity by 2.11‐fold compared to the negative mutant. This study bridges computational design and biocatalytic applications, highlighting the potential of molecular electro‐switches for functional regulation of enzyme.