CHEMICAL MODIFICATION OF DOPAMINE BETA HYDROXYLASE
SAMS, CLARENCE FELTON
Doctor of Philosophy
Dopamine beta hydroxylase (DBH) is the terminal enzyme in the biosynthetic pathway of norepinephrine. The enzyme is a monooxygenase and functions to insert a hydroxyl group on the beta carbon of dopamine to yield norepinephrine. Physical and chemical methods were employed to examine structural features of the enzyme, and the contributions of various amino acids to the catalytic activity were investigated with chemical modifying agents. No free sulfhydryl residues were detected upon titration of DBH with 2-chloromercuri-4-nitrophenol. Reaction with iodoacetamide caused a time dependent loss in activity. The activity change was correlated to the loss of approximately three histidine residues. Methionine reaction was also observed. Reaction of DBH with diethylpyrocarbonate or diazonium tetrazole resulted in the inactivation of the enzyme within fifteen minutes. Modification with either reagent was slightly pH dependent over a range of pH 4.5 to 8.0. Analysis of the diethylpyrocarbonate reacted DBH indicated a loss of one to two histidines per DBH monomer. No tyrosine or lysine modification was observed with this reagent. Inactivation of DBH with diazonium tetrazole resulted in the extensive modification of tyrosine and lysine residues. The activity loss observed with diazonium tetrazole appeared due to general denaturation of the enzyme. Incubation of DBH with trinitrobenzene sulfonic acid, methyl acetimidate or dansyl chloride was carried out to investigate the involvement of lysine residues in enzyme catalysis. Dansyl chloride caused a time dependent loss of activity, completely inactivating the enzyme at a 500 fold excess of reagent over DBH monomers. The other reagents had little effect on DBH activity. DBH was found to bind the fluorescent dyes anilinonaphthalene sulfonate (ANS) and fluorescein. The binding of ANS had no effect on the catalytic competence of DBH, while the binding of fluorescein caused the inactivation of the enzyme. The reaction of DBH with low excesses of fluorescamine resulted in the production of fluorescently labeled active enzyme. No significant difference spectra were observed upon incubation of labeled enzyme with substrates or effector compounds.