Kinetic Mechanism and Intrinsic Rate Constants for the Reaction of a Bacterial Phenylalanine Hydroxylase

• Published in: Biochemistry (Easton) 2016-12, Vol.55 (49), p.6848-6857
• Main Authors: Subedi, Bishnu P, Fitzpatrick, Paul F
• Format: Article
• Language: English
• Subjects: Bacteria - enzymology; Kinetics; Oxygenases - chemistry; Phenylalanine Hydroxylase - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.6b01012

The pterin-dependent aromatic amino acid hydroxylases are non-heme iron enzymes that catalyze the hydroxylation of the aromatic side chain of their respective substrates using an FeIVO intermediate. While the eukaryotic enzymes are homotetramers with complex regulatory properties, bacterial phenylalanine hydroxylases are monomers that lack regulatory domains. As a result, the bacterial enzymes are more tractable for mechanistic studies. Using single turnover methods, the complete kinetic mechanism and intrinsic rate constants for Chromobacterium violaceum phenylalanine hydroxylase have been determined with both tetrahydrobiopterin and 6-methyltetrahyropterin as substrates. In addition the kinetics of formation of the enzyme–pterin complex have been determined with the unreactive 5-deaza, 6-methyltetrahydropterin. For all three pterins, binding of phenylalanine and pterin occurs in random order with binding of the pterin first the preferred pathway. The reaction of the ternary enzyme–phenylalanine–tetrahydropterin complex can be described by a mechanism involving reversible oxygen binding, formation of an early intermediate preceding formation of the FeIVO, and rate-limiting product release.