Effects of sulfhydryl agents on the activation of tryptophan-5-monooxygenase from bovine pineal glands.
Partially purified tryptophan-5-monooxygenase (L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) EC 1.14.16.4)from bovine pineal gland was activated by preincubation with sulfhydryl agents such as dithiothreitol, L-cysteine, cysteamine, L-cysteine ethylester, N-acetyl-L-cysteine, 2-mercaptoethanol and reduced glutathione, at alkaline pH (optimum pH equals 8.5). Dithiothreitol was the most effective of these, leading to approximately 50-fold activation of the enzyme after preincubation. Fe-2+ or other reducing agents such as borohydride, dithionite and ascorbate facilitated the velocity of the activation in the presence of sulfhydryl agents. In the absence of sulfhydryl agents, no activation was observed even in the presence of Fe-2+ or other reducing agents, suggesting an obligatory role or sulhydryl agents during the activation. The relative velocity and full extent of the activation were dependent on the concentrations of both the sulfhydryl agent and the enzyme in the activation mixture. The kinetic analysis of the activation indicated that the sulfhydryl agent reacts with more than 2 sites in the enzyme; one type of site is reduced by sulfhydryl agents, Fe-2+ or other reducing agents and the other specifically modified by a sulfhydryl agent. The activated enzyme did not require any exogenous Fe-2+ for its catalytic activity, but some roles of iron maybe exist in its catalytic reaction. The optimum pH for catalytic reaction of the activated enzyme was approximately 6.5. The apparent Km for L-tryptophan and pteridine cofactor, tetrahydro-pteridine (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin), of the activated enzyme were 30 and 35 muM respectively.
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001894:Borohydrides / Q000494:pharmacology; D002417:Cattle; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D004227:Dithionite / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D010870:Pineal Gland / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014365:Tryptophan Hydroxylase / Q000378:metabolism*
D001894:Borohydrides; D010618:Phenanthrolines; D013439:Sulfhydryl Reagents; D004227:Dithionite; D015082:2,2'-Dipyridyl; D008623:Mercaptoethanol; D007501:Iron; D006899:Mixed Function Oxygenases; D014365:Tryptophan Hydroxylase; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid; D004229:Dithiothreitol