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pubmed:abstractText |
Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine. In the proposed mechanism, a ferryl-oxo species attacks the aromatic ring of tyrosine, forming a cationic intermediate. However, no significant isotope effect is found for wild-type TyrH when 3,5-2H2-tyrosine is used as a substrate. The isotope effect has now been determined with 3,5-2H2-tyrosine using mutant forms of TyrH in which the oxidation of the pterin is uncoupled from hydroxylation of the amino acid. Three mutant enzymes exhibit significant inverse deuterium isotope effects and inverse solvent isotope effects. A proton inventory for the E326A enzyme is consistent with a normal solvent isotope effect of 2.4 on an unproductive step. The results support the proposed mechanism and demonstrate the utility of using mutant proteins with branched pathways to reveal isotope effects which are masked in the wild-type enzyme.
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