Theoretical study of the catalytic mechanism of glyoxylate carboligase and its mutant V51E

Abstract

Glyoxylate carboligase (GCL) catalyzes the ligation of two molecules of glyoxylate to form tartronate semialdehyde (TSA) and carbon dioxide. GCL is unique among ThDP-dependent enzymes because it lacks the canonical glutamate in the active site which is thought to be highly necessary for other ThDP enzymes. In this paper, the catalytic reactions of GCL and its mutant V51E have been explored using a combined QM/MM approach. On the basis of our calculations, the following important points have been obtained: (1) although the glutamate residue is absent in the active site of GCL, the activation of cofactor ThDP is still quite easy with a very low energy barrier of 5.0 kcal/mol; (2) the catalytic cycle can still proceed in the case where no other potential acid–base side chain exists in the active site; (3) for the wild-type GCL, the energy barrier of the most energy-requiring step is 17.9 kcal/mol, which agrees well with the experimental observations; (4) for V51E mutant, the concerted formation of the product TSA and regeneration of the ThDP ylide is calculated to be the rate-limiting step with an energy barrier of 19.5 kcal/mol. It is slightly higher than that of the wild-type GCL (19.5 vs. 17.9 kcal/mol), which can well explain the relatively lower activity of the V51E mutant than the wild-type enzyme. Our results may provide help for further understanding the catalytic mechanism of GCL.