The three-dimensional structure of the enzymes phenylalanine hydroxylase and tryptophan hydroxylase will be studied using crystallographic methods. The structures of the catalytic domain of human phenylalanine hydroxylase, catalytic domain plus tetramerization domain, catalytic domain plus regulatory domain, and catalytic domain with catecholamine inhibitors have previously been determined in my lab at various resolutions (1.9 Angstroms to 3.0 Angstroms). The family of aromatic amino acid hydroxylases are known to be involved in inherited metabolic disorders. The enzyme phenylalanine hydroxylase is the known cause of the disease known as PKU (phenylketonuria). The enzyme normally converts the essential amino acid phenylalanine to tyrosine. Failure of the conversion results in a buildup of phenylalanine. Excessive amounts of phenylalanine are toxic to the central nervous system and causes severe problems associated with PKU. Based on the three-dimensional structure of the modeled full-length enzyme, the mutations will be mapped and site- specific mutagenesis will be conducted to understand the structural basis of PKU. As a complement to the studies on phenylalanine hydroxylase, tryptophan hydroxylase will be studied as comparisons of it and phenylalanine hydroxylase may provide important insights into the mechanisms of PKU. A second goal of the research is to understand the structural basis of pterin-dependent hydroxylase catalysis. The three-dimensional structure of both enzymes will be determined in the presence of co-factors, substrates, and inhibitors in order to understand the mechanism of catalysis and role of inhibitors. The last goal is to crystallize and determine the three-dimensional structure of the intact holoenzyme. By combining the structures of the isolated domains with adequate overlap, a proposed full-length enzyme model can be assembled. However it is important to determine the three dimensional structure of the full-length enzyme in order to fully understand the complex regulatory, catalytic, and stability issues associated with this family of enzymes.
