Formation and Fate of Tyrosine

Abstract

Tyrosine in an hepatocyte is transported from the plasma, synthesized from phenylalanine, or released during protein turnover. Effects of phenylalanine and tyrosine on the formation and fate (partitioning) of tyrosine from the different sources were examined in primary rat hepatocyte cultures. Rates of tyrosine degradation, transport, incorporation into and release from protein, and synthesis from phenylalanine were measured as well as the intracellular dilution of labeled tyrosine and phenylalanine incorporated into protein. We found tyrosine had little effect on phenylalanine hydroxylation over a wide range of conditions, that transported tyrosine and tyrosine from phenylalanine are in different metabolic pools, and that there appears to be channeling of newly synthesized tyrosine during degradation. In addition, under some conditions, intracellular partitioning of tyrosine is determined by tyrosine concentration. Specifically, if extracellular tyrosine is low and phenylalanine is at a normal plasma level, tyrosine use in protein synthesis takes precedence over tyrosine degradation or export. It is proposed that the mechanism controlling this is kinetic, based on relative rates of tyrosyl-tRNA formation and tyrosine degradation and export. A quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes is given, incorporating tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine use and release during protein turnover.In animals, tyrosine is formed by hydroxylation of phenylalanine in a reaction catalyzed by phenylalanine hydroxylase (1, 2). The reaction occurs almost exclusively in liver (3), it is the first step in phenylalanine degradation (1, 4), and phenylalanine appears to be its primary regulator (5-7). Since liver is the site of tyrosine degradation as well as formation, the size of the intracellular tyrosine pool and the relative rates of tyrosine synthesis and degradation might be expected to depend, at least in part, on tyrosine concentration. Tyrosine could directly or indirectly (e.g. through the phenylalanine hydroxylase cofactor tetrahydrobiopterin (1,8)) affect the rate of phenylalanine hydroxylation, it could affect the rate of tyrosine degradation, or it could affect the disposition (metabolic routing) of tyrosine in the cell. The mechanisms are not mutually exclusive, and all could operate, although a direct effect of tyrosine on phenylalanine hydroxylase has not, so far, been observed either in studies with purified enzyme or in the few in situ experiments that have addressed this question (9).Historically, studies of regulation of the tyrosine pool have focused on tyrosine degradation, and most of the interest in degradation has been on control of tyrosine aminotransferase which catalyzes the first step in the pathway, deamination to p-hydroxyphenylpyruvate. The tyrosine aminotransferase reaction, although reversible, is usually considered the rate-limiting and regulated step in tyrosine metabolism (see Refs. 10 -12, but see also ...

Mechanism of Aromatic Amino Acid Hydroxylation

“…Phosphorylation of PheH alters the equilibrium between the open and the closed forms of the enzyme (10)(11)(12), while phosphorylation of TyrH decreases the inhibition by catecholamines (13); the effects of phosphorylation of TrpH have not been elucidated. With both PheH and TyrH, phosphorylation appears to convert the enzymes between active and inactive forms without altering details of substrate binding or the catalytic mechanism.…”

Probing the Role of Crystallographically Defined/Predicted Hinge-bending Regions in the Substrate-induced Global Conformational Transition and Catalytic Activation of Human Phenylalanine Hydroxylase by Single-site Mutagenesis

“…In the present study the biochemical/biophysical properties observed for the described mutant forms of fulllength tetrameric hPAH have given additional information on the relationship between enzyme dynamics/flexibility and catalytic activity (V max ). The catalytic activity of PAH was measured either by varying the L-Phe concentration or the BH 4 concentration. Whereas varying substrate concentration resulted in sigmoid steady-state kinetics, the BH 4 dependency demonstrated classical Michaelis-Menten kinetics, and a higher V max was also obtained for the latter (Table II).…”

“…The tetramer is a dimer of two conformationally different dimers, denoted AC and BD, related by a 2-fold noncrystallographic symmetry axis (3). Based on a number of independent biochemical and biophysical criteria, it is generally accepted that the enzyme exists in at least two different activity and conformational states (4 -8), and that it undergoes a large scale but relatively slow (secondsto-minutes) global conformational transition (isomerization) upon binding of its substrate L-Phe, which is characteristic of a hysteretic enzyme (5)(6)(7)(8). Thus, the catalytic activation of tetrameric hPAH by its substrate can be represented by the equilibria in Equation 1 (6,8),…”

“…that catalyzes the hydroxylation of L-Phe to L-Tyr in the presence of the pterin cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH 4 ) and dioxygen, the rate-limiting step in L-Phe catabolism. Mutations in human PAH (hPAH), leading to altered kinetic properties and/or reduced in vivo stability of the enzyme, are associated with the autosomal recessive metabolic disorder phenylketonuria/hyperphenylalaninemia, and so far 439 putative disease-causing alleles have been identified in the PAH gene coding for a 452-residue polypeptide (1) (www.…”

Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

“…For wild type PheH, the formation of tyrosine from phenylalanine was determined by monitoring the absorbance increase at 275 nm [25]. For the mutant enzymes, a more sensitive HPLC assay [18,26] was used.…”