Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single-Turnover Experiments

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-BH4-phenylalanine complex begins with the rapid binding of BH4 (Kd = 65 µM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (Kd = 130 µM) is approximately ten-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, detectable 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 ten-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.

Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering

“…Instead, the proximity of the N-terminal tail to the BH 4 binding site that was previously observed in the PheH-BH 4 binary complex 32 suggests that its presence may perturb BH 4 binding 31 , which would in turn prevent the conformational changes that are needed to complete the active site pocket 14 , such as the closure of the Tyr138 loop and the loops containing Thr380 and Pro279. An additional clue pointing to BH 4 ’s central role in allosteric activation is the observation of an ordered kinetic mechanism in which BH 4 binds before L-Phe 33 , despite the fact that L-Phe and BH 4 do not interact directly in the pre-catalytic ternary complex 32 . Thus, our structures support a specific order in which allosteric activation must occur: the N-terminal tail first unbinds from the active site to allow for ordered substrate binding, which is accompanied by the movement of the Tyr138 loop and compaction of the catalytic domain to a closed conformation that completes the active site.…”

Structural insights into the regulation of aromatic amino acid hydroxylation

“…Human PheH is reported to have significant activity even in the absence of activation by phenylalanine, but otherwise the regulatory mechanism is assumed to be the same [14]. Mutant forms of rat or human PheH lacking the N-terminal ~117 residue regulatory domain are fully active [12,15], so that the regulatory domain is required to form the low activity resting form of the enzyme. The activated enzyme exhibits positive cooperativity in its kinetics with phenylalanine as a substrate [16], so that the regulatory mechanism may be more complex than indicated by the model in Figure 4.…”

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

“…PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2,3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic.…”