Conformational Properties and Stability of Tyrosine Hydroxylase Studied by Infrared Spectroscopy

Abstract

The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% ␣-helix, 35% ␤-extended structures (including ␤-sheet), 14% ␤-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in ␣-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.Tyrosine hydroxylase (TH, 1 EC 1.14.16.2) is a non-heme iron and tetrahydrobiopterin-dependent enzyme that catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DO-PA), the first and rate-limiting step in the biosynthesis of catecholamines (1, 2). The catalytic activity of TH seems to be short-term regulated by feedback inhibition by catecholamines and by reversal of this inhibition by phosphorylation (1, 3).Until the recent cloning and overexpression of TH from different species (4 -6), the source of the enzyme has mostly been the bovine adrenal medulla or pheochromocytoma (PC12) cells. However, these enzymes are isolated partially inhibited by catecholamines bound at the active site and are activated severalfold by phosphorylation of Ser-40 by the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (7-9). This activation has been related to the increased dissociation rate of catecholamines from the active site on phosphorylation (3).Human TH exists as several different isoforms generated by alternative splicing of pre-mRNA (10, 11); isoform 1 (hTH1) is the most abundant species in the adrenal medulla and in the substantia nigra of the brain (10). When expressed in Escherichia coli, human TH isoforms are isolated as homogeneous tetrameric apoenzymes composed of identical 56-59-kDa subunits (12) that are rapidly activated (up to 40-fold) by binding of 1 atom of Fe(II) per subunit (4). The recombinant human enzymes are inhibited by catecholamines that chelate iron at the active site, and this inhibition is partially reversed by phosphorylation of Ser-40 by cAPK (3, 12, 13).The three-dimensional structure of TH remains to be deter...

Topology of sarcoplasmic reticulum Ca2+‐ATPase: An infrared study of thermal denaturation and limited proteolysis

“…Native sarcoplasmic reticulum has a similar thermal denaturation profile (data not shown) only shifted 3-5°C toward higher temperatures. The assignment of the band at 1,625 cm" to monomermonomer contacts in nondenatured oligomeric proteins is also supported by previous studies of cytochrome oxidase, where the band is present in the oligomeric mitochondrial enzyme but not in the monomeric form from P denitrificans (Haltia et al, 1994), and by the recent data on tyrosine hydroxilase where a correlation has been found between the presence of the 1,625 cm-' band and the monomer-monomer interaction (Martinez et al, 1996).…”

“…This band is different from the one at 1,618 cm", indicative of aggregation, that appears around 55 "C. Bands at such low frequencies are not easily found in nondenatured proteins and have recently been assigned to protein-protein contacts in oligomeric proteins (Martinez et al, 1996). Thus, the thermal profile of this band would indicate that while the protein is oligomeric at room temperature, it becomes transiently a monomer in the process of thermal denaturation.…”

“…4). The spectrum at 52°C shows the 1,655 cm" band as the major component (58%) together with a band at 1,636 cm" (36%) that includes not only the P-sheet structure, but can also overlap with the unordered structure component (Martinez et al, 1996). Note that the bandwidths of the various components at this temperature are increased.…”

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N‐Terminus of the Enzyme

“…Recent physicochemical studies focusing on the redox states of iron incorporated into TH molecule have clarified precisely the mechanisms by which dopamine chelates iron at the active site of the enzyme. In addition, the trapping of the active site iron in the ferric state, the binding mode of 6RBPH4 with iron, and the reduction of ferric iron by 6RBPH4 during catalytic turnover have been extensively characterized (Michaud-Soret et al, 1995;Martinez et al, 1996;Meyer-Klaucke et al, 1996;Ramsey et al, 1996). More recently, the differential binding of dopamine to the ferric and ferrous state TH was clearly demonstrated (Ramsey and Fitzpatrick, 1998).…”

“…The C-domain is located at the C-terminal two-thirds of the molecule and binds the substrates (L-tyrosine and molecular oxygen) and the cofactor (6RBPH,). In contrast, the R-domain has been assigned to the N-terminal end (Hoeldtke and Kaufman, 1977;Abate et al, 1988;Abate and Joh, 1991) and has an important role in substrate specificity and in control of the catalytic activity (Haavik et al, 1990;Daubner et al, 1992;Lohse and Fitzpatrick, 1993;Martinez et al, 1996). Deletion studies have further defined the location of the regulatory domain to the N-terminus.…”