Cellular senescence involves progressive acidification, but how cells sense and adapt to this pH shift remains unclear. Here we report that metabolic enzyme GOT1 functions as a pH sensor that undergoes liquid-liquid phase separation (LLPS) to combat senescence. Proteomic analysis identified GOT1 upregulation in aged human lung cells. Acidic conditions mimicking senescence directly induce GOT1 LLPS via its N-terminal intrinsically disordered region (IDR1), recruiting ME1 to form dynamic enzymatic co-condensates that scavenge reactive oxygen species and alleviate oxidative stress. To quantitatively interrogate GOT1's pH microenvironment during senescence, we engineered BDP-PLP, a first-in-class fluorescent probe conjugating the native GOT1 cofactor pyridoxal phosphate to a BODIPY fluorophore. Operating via a binding-inhibited PET mechanism, this probe enables high-specificity GOT1 targeting and pH-dependent fluorescence lifetime imaging (FLIM). Using FLIM, we achieved quantitative real-time visualization of pH dynamics within GOT1 condensates in living cells, revealing that phase separation generates a highly acidic local microenvironment critical for its anti-senescence function. This study uncovers a pH-triggered phase separation mechanism that bolsters antioxidant defense via metabolic enzyme co-condensation, offering new perspectives on metabolic adaptation in aging and establishing a chemical tool for probing microenvironmental dynamics.
