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 $\text{(6R)-}\text{l}\text{-erythro-1′,2′-}\text{dihydroxypropyltetrahydropterin}$ [$\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$] as cofactor, which is supposed to be a natural cofactor. Two different K_m values for tyrosine, oxygen and natural $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ itself were obtained depending on the concentration of the tetrahydrobiopterin cofactor. In contrast, when unnatural $\text{(6S)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ was used as cofactor, a single K_m value for each tyrosine, oxygen and the cofactor was obtained independent of the cofactor concentration. The lower K_m value for $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ 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 μM with $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ 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 $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ as cofactor.