Phenylalanine Hydroxylase Inactivation in Wildtype and PKU-causing Mutant Enzymes

Gobind Singh
Boston University

Phenylketonuria (PKU) is the most common inborn metabolic genetic disorder of clinical significance. Approximately 1:10,000 individuals suffer from PKU in the US. This enzyme and those mutations that give rise to PKU in the human population can now be readily expressed through the use of recombinant molecular biological techniques using the bacteria Escherichia coli as host. My project involves the use of liquid chromatography/mass spectroscopy to identify the protein side chains that are irreversibly modified during enzyme inactivation. This inactivation process is believed to be the direct consequence of abortive active site chemistry and is much more readily observed in PKU-inducing mutants than in the case of wildtype enzyme. A comparison of the data from normal PAH with inactivated PAH will allow us to determine which residues are modified. The use of structural data will then allow us to place the modification in the three dimensional structure. This is useful for understanding the mechanism of inactivation in PKU-causing mutant enzymes. This deeper understanding of the underlying chemical defects found in PKU-inducing mutants may lead to new approaches in the treatment of this type of disorder.

A second aspect of this project involves studies aimed at observing the intermediate iron-oxo species believed to be responsible for L-phe catalysis. Previous MCD samples showed PAH in an R-state with Fe in both 5 and 6-coordinate geometries when activated with para F-phe. In order to test whether active sites in these MCD samples are fully loaded with para F-Phe, the Km for para F-Phe is needed to analyze the inhibitory properties of this substrate. It has already been determined that para-Fluoro phenylalanine is a weaker substrate than phenylalanine and that although the rate of pterin oxidation in para-F activated PAH is normal, PAH kinetics shift from a sequential to a ping-pong mechanism. With data available from both of these studies, the correct structure of the active site along with the location of the inactive site in PKU-mutant rising enzymes will allow for future studies to be conducted in order to better understand the chemical basis of PKU.