Substituting Tyr¹³⁸ in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation

Abstract

Mammalian phenylalanine hydroxylase ( PAH ) is a key enzyme in l ‐phenylalanine ( l ‐Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate l ‐Phe is the key player in this transition. X‐ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr 138 ‐loop to the active site in the presence of l ‐Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr 138 point mutants. A high linear correlation ( r 2 = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate‐activated full‐length tetramers. In the tetramers, a correlation ( r 2 = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same l ‐Phe concentration. The new data support a similar functional importance of the Tyr 138 ‐loop in the catalytic domain and the full‐length enzyme homotetramer.

Skin transcriptomic correlates of cave-dwelling Sinocyclocheilus cavefish

“…We found that the tyrosine metabolic pathway, influenced by habitat, played a role in determining melanosis in the skin of Sinocyclocheilus. Previous research has demonstrated that decreased phenylalanine-4hydroxylase (PAH, OG: 0000390) activity can affect Try activity (Leandro et al, 2017), and inhibition of phenylalanine conversion to tyrosine will lead to reduced melanin production (Staudigl et al, 2011;Zhou et al, 2021). However, we found low expression of the gene encoding PAH in these species.…”

Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation

“…Nevertheless, residues 176–190 centred around F183 have indeed been shown to have an important catalytic role in TH, controlling the coupling of amino acid hydroxylation to tetrahydropterin cofactor oxidation 47 . In the equivalent Y138-centred active site loop in PAH, which is involved in enzyme activation and catalysis 10 , 48 , large conformational changes effected by ligand binding have also been observed.…”

Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

“…This implies that this residue has a functional importance in TPH1. The importance of this loop and in particular this residue is supported by findings in PAH and TH where the corresponding residues Tyr138 and Phe184 have been found to be important for proper enzymatic function. , …”

“…For PAH, it has been suggested that the closing mechanism might protect the co-substrate to enable a high degree of coupling efficiency. 59 Similarly, the loop in TH has been proposed to exclude water from the active site to avoid reaction of the water with the hydroxylating intermediate before hydroxylation of substrate can occur. 48,60,61 A similar functional importance of the loop in TPH is supported by the very high sequence conservation among vertebrate species observed for both TPH isoforms.…”

“…The importance of this loop and in particular this residue is supported by findings in PAH and TH where the corresponding residues Tyr138 and Phe184 have been found to be important for proper enzymatic function. 59,60 Crystal structures of TPH and PAH have clearly revealed the presence of an open and a closed conformation. For PAH, closed conformations have only been observed when both a pterin and a substrate analogue are bound.…”