Kinetic properties of tyrosine hydroxylase with natural tetrahydrobiopterin as cofactor.

A reproducible purification procedure of native tyrosine hydroxylase (L-tyrosine, tetrahydropteridine : oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the soluble fraction of the bovine adrenal medulla has been established. This procedure accomplished a 90-fold purification with a recovery of 30% of the activity. This purified enzyme served for studying the kinetic properties of tyrosine hydroxylase using (6R)-L-erythro-1',2'-dihydroxypropyltetrahydropterin [(6R)-L-erythro-tetrahydrobiopterin] as cofactor, which is supposed to be a natural cofactor. Two different Km values for tyrosine, oxygen and natural (6R)-L-erythro-tetrahydrobiopterin itself were obtained depending on the concentration of the tetrahydrobiopterin cofactor. In contrast, when unnatural (6S)-L-erythro-tetrahydrobiopterin was used as cofactor, a single Km value for each tyrosine, oxygen and the cofactor was obtained independent of the cofactor concentration. The lower Km value for (6R)-L-erythro-tetrahydrobiopterin was close to the tetrahydrobiopterin concentration in tissue, indicating a high affinity of the enzyme to the natural cofactor under the in vivo conditions. Tyrosine was inhibitory at 100 microM with (6R)-L-erythro-tetrahydrobiopterin as cofactor, and the inhibition by tyrosine was dependent on the concentrations of both pterin cofactor and oxygen. Oxygen at concentrations higher than 4.8% was also inhibitory with (6R)-L-erythro-tetrahydrobiopterin as cofactor.