Tetrahydrobiopterin (BH4) regulates the production of nitric oxide, serotonin, dopamine and tyrosine by serving as an essential cofactor for the enzymes that produce these molecules. The long-range objectives of this research are to understand the mechanisms which regulate GTP cyclohydrolase I (GC), the first and rate-limiting enzyme in the biosynthetic pathway for BH4. Dr. Hatakeyama has focused on investigating the role of a newly identified feedback regulator protein (Frp) in the regulation of GC activity. Frp mediates feedback inhibition of GC activity by BH4. Furthermore, Frp changes the kinetic behavior of GC from sigmoidal to hyperbolic in the presence of phenylalanine, which is the substrate for the BH4-dependent enzyme phenylalanine hydroxylase. GC is a decamer showing a positive cooperativity against GTP. The current working hypothesis is that the catalytic function of GC is determined by different conformational states of the complexes between two Frp molecules (pentamer of 9.5 kDa subunit) and one GC (decamer of 25-kDa subunit) which are formed by the binding of respective effectors. The goal of the proposed research is to define the relationship among the kinetic behavior of GC activity, the effector binding to protein and the protein conformation. The P.I. has established bacterial overexpression systems for both Frp and GC and the purification procedures for each protein.
The specific aims are: 1) to characterize the effects of BH4 and phenylalanine on kinetic parameters of GC and to determine the effectors which modulate the system; 2) to determine the subunit composition of the complexes by gel filtration, to examine the effector binding to the complexes by gel filtration, and to examine the effector binding to the complexes and individual proteins by equilibrium dialysis to determine the affinity, stoichiometry, and specificity of the effectors; 3) to define the gross conformational states of Frp, GC and the complexes in association with the binding of effectors by sedimentation and protease sensitivity; and 4) to determine the three-dimensional structure of Frp by crystallography. Crystals of Frp that diffract to 2.0 Angstroms have been obtained. These studies are fundamental for understanding the molecular mechanisms of allosteric regulation of GS by Frp and will provide insights into the regulation of the biosynthesis of BH4. A better understanding of the regulation of BH4 biosynthesis is expected to yield insight into the altered metabolism of BH4 reported in some types of phenylketonuria, Parkinson s disease, familial dystonia and infectious diseases.
