期刊
BIOCHEMISTRY
卷 50, 期 11, 页码 1928-1933出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi1019868
关键词
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资金
- National Institutes of Health [R01 GM047291, R01 GM 69657]
- Welch Foundation [AQ-1245]
- National Science Foundation [CHE-724084, MCB-642058]
Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mossbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-Fe-57(II) phenylalanine-6-methyltetrahydropterin complex with O-2 reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mossbauer parameters of the intermediate (delta = 0.28 mm/s, and |Delta E-Q| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme substrate complex reacts with oxygen to form an Fe(IV)=O intermediate with a rate constant of 19 mM(-1) s(-1), the Fe(IV)=O intermediate hydroxylates phenylalanine with a rate constant of 42 s(-1), and rate-limiting product release occurs with a rate constant of 6 s(-1) at 5 degrees C.
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