Biotin protein ligases constitute a family of enzymes that catalyze the linkage of biotin to biotin-dependent carboxylases. In bacteria, these enzymes are functionally divided into two classes: the monofunctional enzymes that catalyze only biotin addition and the bifunctional enzymes that also bind to DNA to regulate transcription initiation. 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. Included among these properties are the order of substrate binding and linkage between the oligomeric state and ligand binding. To test this hypothesized relationship between functionality and biochemical properties in ligases, we have conducted studies of the 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. Measurements performed using isothermal titration calorimetry and fluorescence spectroscopy indicate that, in contrast to the bifunctional E. coli enzyme, substrate binding does not occur by an obligatorily ordered mechanism. Finally, thermodynamic signatures of ligand binding to the monofunctional enzyme differ significantly from those measured for the bifunctional enzyme. These results indicate a correlation between the functional complexity of biotin protein ligases and their detailed biochemical characteristics.