Mitochondrial pyruvate dehydrogenase complex (PDHC) is an enzyme vital for glucose and intermediary metabolism. PDHC is a thiaminedependent enzyme whose low activity is implicated in several neurodegenerative diseases including congenitial lactic acidosis. PDHC has been shown to be inactivated by PDHC-specific kinase (PDK) and reactivated by PDHC phophatase. The effect of one of the natural mutations, H263L in the Ela subunit of PDHC, found in some patients suffering from lactic acidosis, was investigated by homology modeling and site-directed mutagenesis. A 3dimensional structure for E10 and E13 subunits (wild type) was generated based on the crystal structure of transketolase using a homology molecular modeling program. This structure suggested that His 263 is involved in thiamine pyrophosphate (TPP) binding. Our biochemical studies using recombinant E10 produced via a co-expression of El and El, subunits showed that TPP binding inhibits the phosphorylation by recombinant PDK. These studies were further complemented by generating the mutant H263L by site-directed mutagenesis. The gene for H263L was subcloned into an expression system which also contained the gene for El subunit. The activity of partially purified mutant El and E1B from this co-expression system was found to be 4% of that for the wild type. More biochemical and molecular modeling approaches are underway to determine the binding constants for TPP with H263L mutant and also to determine whether this mutation causes the PDH protein more vulnerable to phosphorylation relative to the wild type.