Kinetic mechanism of phenylalanine hydroxylase: Intrinsic binding and rate constants from single-turnover experiments

Kenneth M. Roberts, Jorge Alex Pavon, Paul F. Fitzpatrick

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH4) and O2. A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH 4-phenylalanine complex begins with the rapid binding of BH 4 (Kd = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (Kd = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O2 rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s-1. Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines kcat. Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

Original languageEnglish (US)
Pages (from-to)1062-1073
Number of pages12
JournalBiochemistry
Volume52
Issue number6
DOIs
StatePublished - Feb 12 2013

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Kinetic mechanism of phenylalanine hydroxylase: Intrinsic binding and rate constants from single-turnover experiments'. Together they form a unique fingerprint.

  • Cite this