Galactokinase 1 Positively Regulates Mitochondrial Respiration by Phosphorylating TIMM13 as a Protein Kinase

ABSTRACT Galactokinase 1 (GALK1) is a metabolic kinase that phosphorylates galactose. Mutations in GALK1 cause type II galactosemia, which manifests as cataracts and extra‐ocular symptoms. The mechanisms underlying these symptoms are not fully understood. In this study, we explored a novel function of GALK1 beyond galactose phosphorylation. GALK1 is ubiquitously expressed across human tissues, regardless of developmental stage. GALK1 phosphorylates TIMM13 at the Y73 residue. The phosphorylated Y73 stabilizes TIMM13 by electrostatically interacting with two cationic residues, thereby enhancing its affinity for Zn 2 + . This structural change ensures the mitochondrial translocation of TIMM13 in the correct conformation. Disruption of Y73 phosphorylation impairs mitochondrial bioenergetics and dissipates the mitochondrial membrane potential. Furthermore, galactose competitively inhibits GALK1‐mediated phosphorylation, suggesting that mitochondrial activity is modulated by carbohydrate availability. Galactosemia‐associated GALK1 mutations showed compromised activity in TIMM13 phosphorylation, subsequently repressing mitochondrial respiration. Collectively, the GALK1‐TIMM13 axis appears to provide a potential link between galactose metabolism and mitochondrial respiration.