Protein-enhanced decarboxylation of the covalent intermediate in benzoylformate decarboxylase—Desolvation or acid catalysis?

Benzoylformate decarboxylase (BFD) enhances the rate of decarboxylation of its key intermediate compared to the nonenzymic reaction by a factor of about 106. It has been proposed that desolvation into a hydrophobic environment will lower the reaction barrier in TDP-dependent decarboxylases. The competition of thiamin thiazolone diphosphate (TTDP) with the cofactor thiamin diphosphate (TDP) provides a dynamic indication of the relative hydrophobicity of the cofactor binding site. BFD binds the more polar TDP tightly in the presence of excess TTDP. Therefore, desolvation would not be likely to occur during catalysis. Unlike TDP enzymes that have electron acceptors as substrates, decarboxylases require protonation to produce the precursor to the aldehyde product. A mechanism involving an associated acid that traps the carbanion generated upon C–C bond breaking will permit diffusional separation of carbon dioxide and generate the appropriate precursor to the product aldehdye. This would also account for avoidance of a competitive reaction. Hasson’s detailed structure of BFD shows a highly polar active site with histidines in the vicinity of the substrate. Reports of a reduction of kcat to near the nonenzymic rate without a large effect on Km upon specific replacement of these histidines with alanine fit this alternative. In TDP enzymes involving oxidation or condensation, an electrophilic substrate or second cofactor will be bound (and no proton will be required). This will acquire the electron density of the carbanion itself. In such cases, protonated side chains are not functional while hydrophobic environments would promote the internal transfer.

Figure optionsDownload as PowerPoint slide