Correlation Between Functionality and Biochemical Properties in Biotin Protein Ligases

Abstract

Biotin protein ligases are a family of enzymes that catalyze biotin linkage to biotin-dependent carboxylases. In microorganisms these enzymes are functionally divided into two classes including the monofunctional class that only catalyzes biotin addition and the bifunctional class that also binds to DNA to regulate transcription. Biochemical and biophysical studies of the bifunctional Escherichia coli ligase suggest that several properties of the enzyme have evolved to support its additional regulatory role. These properties include the order of binding of multiple substrates and linkage between oligomeric state and ligand binding.In order to test the hypothesized relationship between bifunctionality and enzymatic properties in ligases, we have carried out studies of monofunctional ligase from Pyrococcus horikoshii. Sedimentation equilibrium measurements to determine the effect of ligand binding on oligomerization indicate that the enzyme exists as a dimer regardless of liganded state. Isothermal titration calorimetry and fluorescence spectroscopy measurements of substrate binding indicate that, unlike in the E. coli enzyme, substrate binding is not ordered. Finally, thermodynamic signatures of ligand binding to the monofunctional enzyme differ significantly from those measured for the bifunctional enzyme. Combined studies of the bifunctional and monofunctional biotin ligases indicate a link between the functionality of these enzymes and their detailed biochemical characteristics.