Chemical modification of dopamine beta-hydroxylase.
Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002730:Chloromercurinitrophenols; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004299:Dopamine beta-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D005450:Fluorescamine / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D013438:Sulfhydryl Compounds / Q000032:analysis; D013777:Tetrazoles / Q000494:pharmacology
D000596:Amino Acids; D002730:Chloromercurinitrophenols; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D013777:Tetrazoles; C007314:5-diazo-1H-tetrazole; C026149:2-chloromercuri-4-nitrophenol; D005450:Fluorescamine; D004299:Dopamine beta-Hydroxylase; D004047:Diethyl Pyrocarbonate; D019807:Iodoacetic Acid; D007460:Iodoacetamide