Mutagenesis studies revealing the complementary roles of effector protein binding and the hydroxylase active site in catalysis by toluene 4 -monooxygenase

Toluene 4-monooxygenase (T4MO) is a multi-component enzyme that catalyzes the regiospecific hydroxylation of toluene. The results described in this thesis illustrate how two of the proteins from this complex, the effector protein and hydroxylase, work in concert to define the observed catalytic properties of the enzyme. Mutagenesis and screening strategies were used to identify the roles of specific residues in catalysis and regiospecificity. The conserved active site threonine, T201, was found to have no effect on steady-state catalysis by T4MO. Putative active site residues were identified that, when mutated, resulted in dramatic changes in regiospecificity without deleteriously affecting the catalytic competency of the enzyme. The location of these residues was identified using a three-dimensional homology model. The mutagenesis and screening experiments, in conjunction with the homology model, identified a ring of amino acids surrounding the diiron cofactor that appear to be critical in defining the regiospecificity of the enzyme, suggesting the global structure of the active site defines the observed regiospecificity. Kinetic studies using substoichiometric amounts of effector protein, alternative substrates, and hydroxylase mutants have shown the primary role of the effector protein is to enhance catalytic activity, while reinforcing the hypothesis that the architecture of the hydroxylase active site is the primary determinant of the regiospecificity. The mechanism of aromatic hydroxylation was investigated using various hydroxylase isoforms in conjunction with deuterated toluene analogs. The intramolecular isotope effect and deuterium retention patterns are consistent with an arene oxide intermediate in the aromatic hydroxylation of toluene catalyzed by T4MO. A conserved structural feature of effector proteins from diiron monooxygenase enzymes, the disordered N-terminal region, was deleted from the T4MO effector protein to determine if it is an essential feature of the protein. Although the deletion increased the apparent K M and decreased the apparent Vmax of the protein, the region was not necessary for catalysis. The combined results of the experiments presented in this thesis led to the development of a model of catalysis by T4MO that illustrates how effector protein binding and the active site architecture may work in concert to mediate the efficient, regiospecific hydroxylation of toluene.