Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogues.
Tyrosine hydroxylase catalyzes the formation of dihydroxyphenylalanine from tyrosine, utilizing a tetrahydropterin and molecular oxygen as cosubstrates. Several approaches were taken to examining the identity of the rate-limiting step in catalysis. Steady-state kinetic parameters were determined with a series of ring-substituted phenylalanines. The Vmax value was unchanged with substrates ranging in reactivity from tyrosine to 4-fluorophenylalanine. Neither 4-pyridylalanine N-oxide, a model of tyrosine phenoxide, nor 4-hydroxy-3-pyridylalanine N-oxide or alpha-amino-3-hydroxy-4-pyridone-1- propionic acid, models of a hydroxycyclohexadienone intermediate, was an effective inhibitor. There was no solvent isotope effect on either the Vmax or the V/KTyr value. These results establish that no chemistry occurs at the amino acid in the rate-limiting step and no exchangeable proton is in flight in the rate-limiting step. The results are consistent with a model in which the slow step in catalysis is formation of the hydroxylating intermediate.
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
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Fitzpatrick
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D002417:Cattle; D007700:Kinetics; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors; D011485:Protein Binding; D012997:Solvents; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D012997:Solvents; D014443:Tyrosine; D010649:Phenylalanine; D014446:Tyrosine 3-Monooxygenase