Tyrosine hydroxylase, an iron-containing, pterin-dependent monoxygenase catalyze the rate-controlling step in the biosynthesis of catecholamine neurotransmitters, the hydroxlation of tyrosine to form dihydroxyphenylalanine. The pterin-dependent monooxygenases are a poorly understood group of enzymes. The primary goal of this research is to understand the catalytic mechanism of tyrosine hydroxylase. The DNA for rat PC 12 tyrosine hydroxylase has been cloned and will be expressed in a high efficiency baculovirus expression system in order to obtain enough pure material for detailed mechanistic studies. The kinetic mechanism will be determined using steady state kinetics and isotope trapping. Rapid reaction techniques will be used to measure the rates of formation of catalytic intermediates. Several nonphysiological amino acids appear to act as substrates for tyrosine hydroxylase; these reactions will be studies to gain information about the identity of the oxygenating species. This class of enzymes, the monooxygenases, catalyze some of the most difficult reactions in biochemistry. Consequently, they have been the objects of a great deal of study. The mechanism of phenylalanine hydroxylase has been well studied in contrast to tyrosine hydroxylase. It shows a number of significant differences; phenylalanine does not appear to be a substrate for tyrosine hydroxylase although the data is not consistent; the redox state of the resting enzymes are different; tyrosine hydroxylase can use H2 02 in place of tetrahydropterins. This study could also offer fundamental insights into the mechanism of other classes of monooxygenases since it has features in common with both flavoprotein hydroxylases and Fe(11) monooxygenases.
