# An Alternative Mechanism of Glutamate Dehydrogenase Inhibition by EGCG: Promotion of Protein Degradation

## TL;DR

This study shows that EGCG can break down GDH proteins, offering a new treatment strategy for diseases caused by hyperactive GDH.

## Contribution

EGCG promotes GDH degradation by forming complexes with trypsin, providing a novel therapeutic approach for HHS.

## Key findings

- The GDH454 mutant has weaker enzymatic activity but greater resistance to trypsin hydrolysis than wild-type GDH.
- EGCG breaks down GDH hexamers into monomers and enhances degradation via trypsin complexes.
- EGCG shows good affinity for both wild-type and mutant GDH proteins, promoting their degradation.

## Abstract

Backgroud: Glutamate dehydrogenase (GDH) is involved in the metabolism of glutamate and ammonia. It is regulated by multiple ligand variants, and hyper-active GDH mutants have been reported for hyperinsulinism hyperammonemia syndrome (HHS). Methods: Here, we constructed the wild-type human GDH and three human GDH454 mutants and investigated their degradation activity and performance under different GDH inhibitors. Results: Protein activity test and SDS-PAGE analysis of the purified proteins showed that the GDH454 mutant from HHS has weaker GDH enzymatic activity but greater resistance to trypsin hydrolysis than the wild type. Interestingly, using the biomolecular interactions technique, it showed that the GDH454 mutant has 109 times weaker affinity for trypsin and 10-fold weaker for epigallocatechin gallate (EGCG) than the wild-type GDH. Subsequently, native-PAGE gel analysis demonstrated that EGCG could break down the GDH hexamer into monomers and form a complex with trypsin to enhance the degradation of both types of GDH. Conclusions: EGCG showed good affinity to both the wild-type and the mutant GDH proteins, promoting protein degradation; this provides a new strategy for the treatment of HHS and other hyper-active GDH-related diseases.