Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-l imiting enzyme in the biosynthesis of catecholamines; its activity is regul ated by feedback inhibition by catecholamine products including dopamine. T o investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, a nd 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in E scherichia coli and purified as a tetrameric form by affinity and size-excl usion chromatography, The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant non fused form. Although maximum velocities of all N-terminus-deleted forms wer e about one-fourth of the wild-type value, there was no difference in Micha elis constants for L-tyrosine or (6R)-(L-erythro-1',2'-dihydroxypropyl)-2-a mino-4-hydroxy-5,6,7,8-tetrahydropteridine (6RBPH(4)) among the four enzyme s. The iron contents incorporated into the three N-terminus-deleted mutants were significantly lower than that of wild type, However, there was no sub stantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type o r del-35 TH form. Dopamine bound to the del-38 more than to the del-35 TH f orm. However, when incubation with dopamine was followed by further inhibit ion with the cofactor 6RBPH(4) dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the am ino acid sequence Gly(36)-Arg(37)-Arg(38) plays a key role in determining t he competition between dopamine and 6RBPH(4) and affects the efficiency of dopamine inhibition of the catalytic activity.