p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-containing enzyme that catalyzes the hydroxylation of activated aromatic compounds. This complex multi-step process appears to be regulated in part by enzymatic conformational changes. The movement of the isoalloxazine moiety of the FAD, recently identified by x-ray crystallography, is one such conformational movement. The crystallographicaly observed flavin position is identified by the presence of substrate-flavin hydrogen bonds. Additionally, the behavior of site directed mutants indicates that specific amino acid residues influence the flavin conformation. We are interested in defining the energetic factors influencing the conformational state of the enzyme in terms of protein-flavin, protein-ligand, and flavin-ligand interactions. This will be accomplished by direct calorimetric measurements of substrate binding to both WT PHBH and site directed mutants, establishing enthalpies and entropies for the formation of enzyme-substrate complexes of known structure. Furthermore, preliminary spectral studies indicate that the flavin position is influenced by temperature. Thus it will also be of interest to measure, by differential scanning calorimetry, the heat capacities of wild-type and mutant enzymes, both free and complexed with ligands, and compare these values to values for apoenzyme. In this way, the contribution of the flavin conformational change to the heat capacity will be discerned.
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