Dimethyl Sulfoxide → L-Tyrosine

Participants

Reaction Steps

PAH activity was measured at 25° C. for 1 min essentially as described (BjØrgo E, et al., Eur. J. Biochem. 1988; 257(1):1-10). Tetrameric hPAH (1-2 μg) was incubated in 20 mM Hepes 0.2 M NaCl pH 7.0, containing catalase (0.04 mg/ml), 0.1-1 mM L-Phe, DMSO 2.5% and 0-100 μM of each compound. After 4 min incubation at 25° C., ferrous ammonium sulphate (100 μM) was added, and the reaction was triggered after 1 min by adding BH4 (10-200 μM) in the presence of 5 mM DTT. In some experiments, the preincubation step with L-Phe was omitted and L-Phe was added together with BH4 to analyze the L-Phe induced activation of the enzyme. To determine the kinetic parameters for BH4 and L-Phe, BH4 was used at 0-200 μM (at fixed 1 mM L-Phe) and L-Phe at 0-1 mM (BH4 was fixed at 75 μM). L-Tyr formed was quantified by HPLC and fluorimetric detection (DØskeland A P, et al., J. Biol. Chem. 1984; 259:11242-11248). The saturation curves were fitted to hyperbolic (for BH4) or sigmoidal (for L-Phe) kinetic models by using Sigma Plot v.9.0 (SPSS Inc).

Provenance & Evidence