Steady-state kinetic mechanism of rat tyrosine hydroxylase.
The steady-state kinetic mechanism for rat tyrosine hydroxylase has been determined by using recombinant enzyme expressed in insect tissue culture cells. Variation of any two of the three substrates, tyrosine, 6-methyltetrahydropterin, and oxygen, together at nonsaturating concentrations of the third gives a pattern of intersecting lines in a double-reciprocal plot. Varying tyrosine and oxygen together results in a rapid equilibrium pattern, while the other substrate pairs both fit a sequential mechanism. When tyrosine and 6-methyltetrahydropterin are varied at a fixed ratio at different oxygen concentrations, the intercept replot is linear and the slope replot is nonlinear with a zero intercept, consistent with rapid equilibrium binding of oxygen. All the replots when oxygen is varied in a fixed ratio with either tyrosine or 6-methyltetrahydropterin are nonlinear with finite intercepts. 6-Methyl-7,8-dihydropterin and norepinephrine are competitive inhibitors versus 6-methyltetrahydropterin and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine, a competitive inhibitor versus tyrosine, shows uncompetitive inhibition versus 6-methyltetrahydropterin. At high concentrations, tyrosine is a competitive inhibitor versus 6-methyltetrahydropterin. These results are consistent with an ordered kinetic mechanism with the order of binding being 6-methyltetrahydropterin, oxygen, and tyrosine and with formation of a dead-end enzyme-tyrosine complex. There is no significant primary kinetic isotope effect on the V/K values or on the Vmax value with [3,5-2H2]tyrosine as substrate. No burst of dihydroxyphenylalanine production is seen during the first turnover. These results rule out product release and carbon-hydrogen bond cleavage as rate-limiting steps.
Fitzpatrick
P F
PF
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011622:Pterins / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000493:pharmacokinetics*
D011622:Pterins; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; C017523:6-methyltetrahydropterin; D014446:Tyrosine 3-Monooxygenase; D010100:Oxygen; D009638:Norepinephrine