Adenosylcobalamin-dependent glutamate mutase: Pre-steady-state kinetic methods for investigating reaction mechanism

Abstract

This chapter shows that pre-steady-state kinetic studies have proved useful in investigating the chemical mechanism of the unusual rearrangement reaction catalyzed by glutamate mutase and have provided insights into the energetics of free radical formation. It is evident that one strategy employed by the protein is to couple an energetically unfavorable step such as Co-C bond homolysis to the formation of more stable substrate-based radicals. Nevertheless, the details of how the protein catalyzes homolysis of AdoCbl and stabilizes the substrate radicals to such a remarkable degree remain unclear. Now that the free energy profile and the crystal structure have been determined for the wild-type enzyme, the stage is set to address this question. The contribution of individual amino acid residues to catalysis can now be dissected with some precision, as the effect of mutations on both the structure of the protein and the free energy profile of the reaction may be determined.