Abstract

Enolase, a dimeric enzyme, interconverts 2-phosphoglyceric acid to phosphoenolpyruvate in glycolysis. Due to its catalytic significance, deficiency in this pathway is rare. In year 2000, a male patient was identified with the first case of [beta][beta]-enolase deficiency. It was later determined that his [beta]-enolase gene (EN03) carried two missense mutations changing two conserved amino acids: G156D and G374E. The authors who identified this disease suggested that the stability of the enzyme might have been altered. Our research focuses on one of the mutations (G374E), which was introduced into yeast enolase. The yeast enzyme was utilized due to its high sequence identity with the human enzyme, multiple known crystal structures bearing conserved structural characteristics and easy laboratory manipulation. The purified G376E protein is folded but nearly inactive. The subunit dissociation constant was increased by 1000-fold. Although the chemical and thermal stability of the enzyme was not greatly altered, the susceptibility towards proteolytic digestion was significantly high due to weaker dimer stability. Furthermore, our kinetic study shows greatly altered catalytic ability and affinity towards metal ion and substrate. Aside from the original mutation, smaller polar and/or charged amino acids were substituted to examine the cause of the changes observed above. The results indicate that although not directly participating in catalysis or subunit interaction, amino acid 376 plays an important role in subunit interaction and catalytic activity