Abstract
Pheochromocytoma tyrosine hydroxylase was reported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the apparent inability to hydroxylate phenylalanine and an unprecedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The purified tumor enzyme was entirely dependent on tetrahydrobiopterin (BH4) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hydrogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (S0.5 Tyr = 15 microM) and BH4 (apparent Km BH4 = 210 microM). In addition, the enzyme catalyzed the hydroxylation of substantial amounts of phenylalanine to tyrosine and 3,4-dihydroxyphenylalanine (apparent Km Phe = 100 microM). Phenylalanine did not inhibit the enzyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations greater than or equal to 50 microM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or exceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hydroxylase from other tissues.
Highlights
THEJOURNAOFLBIOLOGICAL CHEMISTRVVol 266,No 24, Issue of August 25, pp. 16207-16211,1991 Printed in U.S.A. The Hydroxylation of Phenylalanine and Tyrosine by Tyrosine Hydroxylase from Cultured Pheochromocytoma Cells*
Pheochromocytoma tyrosinehydroxylasewas re- Subsequently, using highly purified tyrosine hydroxylase from portedtohaveunusualcatalyticproperties, which bovine adrenal medulla, Shiman et al (3) showed that the might be unique to the tumoenr zyme
The purified PC12 enzyme was assayed for tyrosine hydroxylating activity at 25 “C and in 20 mM Tris-HC1, pH 8.2, such as described by Dix et al (12)
Summary
Vol 266,No 24, Issue of August 25, pp. 16207-16211,1991 Printed in U.S.A. The Hydroxylation of Phenylalanine and Tyrosine by Tyrosine Hydroxylase from Cultured Pheochromocytoma Cells*. Incontrast to tyrosine, the tumor enzyme has the same substrate specificity which showed typical kinetic properties (13, 14), phenylalaand sensitivity to hydrogen peroxide as tyrosine hy- nine was reported to be inactive as a substrate for the PC12 droxylase from othertissues. Ikeda and coworkers (1)reported that crude preparations of rat striatalor adrenal tyrosine hydroxylase catalyzed the hydroxylation of small amountsof phenylalanine. These authors furtherdemonstrated that thereaction was not mediated by a liver type of phenylalanine hydroxylase but was catalyzed by a tyrosine hydroxylase which converted phenylalanine to Dopa’ (2). The findings of Dix and co-workers (12) raise the interesting possibility that tyrosine hydroxylase from tumor cells has unique characteristics of substrate specificity and cofactor utilization, presumably reflecting a change in the structure of the enzyme. BH,, (6R,6S)-5,6,7,8-tetrahydrobiopterin6;-MPH,, 6-methyltetra- Materials-Phenylalanine, L-tyrosine, ~-3,4-dihydroxyphenylhydropterin; SDS, sodium dodecyl sulfate
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