characterization of the catalytic domain of bovine adrenal tyrosine hydroxylase

Vol. 151, No. 3,1988

March 30,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1446-1453

CHARACTERIZATION OF THE CATALYTIC DOMAIN

OF BOVINE ADRENAL TYROSINE HYDROXYLASE

* 1 Smith # 2 Joh* Cory Abate ' , John A. ' , and Tong H. ,1,+

*Laboratory of Molecular Neurobiology

Cornell University Medical College

Burke Rehabilitation Center

White Plains, New York

#Departments of Molecular Biology and Pathology

Massachusetts General Hospital

and Department of Pathology

Harvard Medical School

Boston, Massachusetts

Received February 29, 1988

Mild trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34 kDa fragment which is catalytically active. To determine the structure of the trypsin-digested tyrosine hydroxylase (tTH) relative to the native enzyme and to regulatory phosphorylation sites, bovine adrenal tTH was purified to homogeneity and the sequence of 17 amino acids from the N- terminus was determined. These data indicate that the N-terminus of tTH corresponds to amino acid 158. Thus the catalytic region is contained within the central region of enzyme approximately 17 kDa from the N-terminal and 5 kDa from the C-terminal and does not include phosphorylation sites located in the N-terminus. This

1 Supported by NIMH grant, MH 24285.

2 Supported by a grant from Hoechst Aktiengesellschaft, Federal Republic of Germany.

Abbreviations used were; TH, tyrosine hydroxylase; tTH, trypsin-digested tyrosine hydroxylase; PAIl, phenylalanine hydroxylase; TPH, tryptophan hydroxylase; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DOPA, 3,4,-Dihydroxyphenylalanine.

+To whom correspondence should be addressed.

ooo6-291x/88 $1.5o Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 1446

Vol. 151, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

region of TH shares a high degree of homology with phenylalanine hydroxylase and tryptophan hydroxylase and thus reflects a selective conservation of regions required for catalysis in contrast to the non-homologous regulatory sites. Activation by proteolysis corresponds to an increase in affinity for both substrate and cofactor indicating that the region removed by proteolysis imposes additional constraints on substrate and cofactor binding. These data are consistent with the model that the catalytic core of TH is contained within a 34 kDa region in the highly conserved central portion of the molecule whereas the non-homologous N-terminus regulates cofactor binding and directs substrate specificity. ©198~Acad~icP ..... Inc.

Tyrosine hydroxylase (TH) (tyrosine 3-monooxygenase EC

1.14.16.2) is a pteridine-requiring monooxygenase that catalyzes

the first and rate-limiting step in catecholamine biosynthesis.

In cells of the nervous system and adrenal medulla that produce

these catecholamines, phosphorylation is the major mechanism of

regulation of enzymatic activity in response to short-term

physiological stimulation (c.f. ref i). In vitro, this activation

is mediated by an increase in affinity for pteridine cofactor

with no change in affinity for substrate tyrosine.

In addition, TH can be activated in vitro by trypsin

proteolysis (2,3). This treatment produces a 34 kDa fragment from

the 60 kDa monomer. The trypsin-digested TH (tTH) is

catalytically active and thus contains the catalytic domain of

the enzyme. The 34 kDa fragment, however, cannot be further

activated by phosphorylation (2) suggesting that proteolysis

removes a region of the enzyme which contains the phosphorylation

sites. These observations are consistent with the model (4,5)

that the TH monomer is composed of two functional regions which

are separable by proteolysis: a catalytic domain corresponding to

tTH and a regulatory region which presumably contains the

phosphorylation sites. Furthermore, based on comparison of the

eDNA sequences, it has been proposed (4,5) that the catalytic

domain of TH corresponds to the region which shares a high degree

of homology with the related aromatic amino acid monooxygenases,

phenylalanine hydroxylase (PAH) (5,6) and tryptophan hydroxylase

(TPH) (7) and that this homology reflects a conservation of

sequences required for catalysis. In contrast, it has been

proposed that the non-homologous N-terminal region which contains

the phosphorylation sites (8) is involved in the regulation of

enzyme activity and in directing substrate specificity. Although

the sequence of the phosphorylation sites of TH (8) and PAH (9)

1447


have been reported, this model remains unconfirmed, since the

structure of tTH has not been identified nor have the sequences

of the catalytically regions of either PAH or TPH been

determined.

We sought to investigate the structural and functional

characteristics of tTH with respect to the native enzyme. Toward

this aim, bovine adrenal tTH was purified to homogeneity and its

N-terminal amino acid sequence was determined. The mechanism of

activation of TH by proteolysis was investigated by kinetic

analysis. A preliminary account of this work has been presented

elsewhere (i0). MATERIALS AND METHODS

L-[U-14C] tyrosine (500 mCi/mol) and Silver stain Detection System were from New England Nuclear. Electrophoretic grade acrylamide and SDS and molecular weight standards were purchased from Bio-Rad. Bovine pancreas trypsin (type III) was from sigma. 6-methyl 5,6,7,8,tetrahydropterin (6MPH4) was from Calbiochem- Behring. Native bovine adrenal TH was partially purified as previously reported (ii) up to the first DEAE-cellulose column. Tyrosine hydroxylase activity was determined by the method of Coyle (12) as modified by Reis et al. (13). One unit of activity corresponds to 1 nmol DOPA/ mg/ 7 min. Protein concentration was determined by the method of Lowry (14).

Trypsin-digested tyrosine hydroxylase was isolated from bovine adrenal medulla as previously described (ll).Briefly, tissue was homogenized in 3 vol of potassium phosphate buffer, pH 7.0 (buffer A) containing 0.3 M sucrose and centrifuged at i00,000 x g for 1 h at 4°C. The pellets were resuspended in buffer A and digested with trypsin (0.5 mg/ml) for 15 min at 30°C. Digestion was stopped by the addition of 4-fold excess soybean trypsin inhibitor and centrifuged as above. The tTH was purified from the supernatant as described (ii) up to the second DEAE-cellulose column.

For N-terminal sequencing, tTH was further purified by preparative SDS-PAGE. i00 - 120 ug of protein was loaded on 10% gels and electrophoresis preformed according to the method of Laemmli (15). Protein bands were visualized by the sodium acetate shadowing technique described by Higgins and Dahmus (16). Bands corresponding to tTH were identified by comparison with MW standards. The protein was electroeluted overnight in 50 mM ammonium bicarbonate containing 0.1% SDS using an ISCO model 1750 electroconcentration cell (3 watts, constant power). Protein was recovered by precipitation with 4 volumes acetone:methanol (i:i) and dissolved in HPLC-grade water containing 0.05% SDS. Purity of the sample was assessed by SDS-PAGE using silver stain detection as described by the instructions of the manufacturer (New England Nuclear); the protein concentration was estimated by amino acid analysis using a Beckman 6300 Amino Acid Analyzer.

Protein sequence analysis of 200 pmols of the purified tTH was carried out with an Applied Biosystems 470A Protein Sequencer and a 120A Pth Analyzer.

Michaelis constants of native and trypsin-digested bovine TH were determined by Lineweaver-Burke kinetic analysis. Data was analyzed by linear regression analysis.

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RESULTS AND DISCUSSION

Bovine adrenal tTH was purified to apparent homogeneity by

preparative SDS-PAGE (Figure i). Since the protein was isolated

from denaturing gels, it was not enzymatically active. However,

the 34 kDa protein, which was readily identified as the major

band in the preparation, comigrated with enzymatically active tTH

when run in parallel on SDS gels and was immunoreactive with tTH

antisera on Western blots (not shown).

The purified protein was subjected to protein sequence

analysis and the sequence of 17 amino acids was determined (Table

I). The N-terminus of bovine tTH corresponded to position 158

(Figure 2A) when compared to the complete sequence of rat TH

deduced from a cDNA (4); an arginine at position 157 was

presumably the N-terminal trypsin digestion site. In addition,

this is the first available sequence for bovine TH. For the

sequences compared, the bovine and rat enzymes are 80%

homologous. However, these data are clearly too limited to infer

homology between the species. Such analysis awaits more extensive

sequencing of the bovine enzyme or cDNA.

?ii

2 ] Figure i: SDS-PAGE of purified bovine adrenal trypsln-digested tyrosine hydroxylase (tTH). Electrophoresis was preformed according to the method of Laemmli (15) using a 10% gel. Protein bands were visualized by silver staining (New England Nuclear). Molecular weight (MW) standards were soybean trypsin inhibitor (21,500 Da), carbonic anhydrase (31,000 Da), ovalbumin (45,000 Da) bovine serum albumin (66,200 Da), and phosphorylase B (92,500 Da). Lane i, MW standards, 200 ng each; lane 2, tTH, 150 rig.

1449


Table I Sequence data of bovine trypsin-digested tyrosine hydroxylase (tTH). The sequence was determined for approximately 200 pmols of purified tTH by automated Edman degradation using an Applied Biosystems 470A Protein Sequencer and a 120A Pth Analyzer. The repetitive yield was calculated from yields of representative, stable, Pth-amino acids.

CYCLE A.A PMOL

i ------

2 ------

3 (Gly) 406 4 GIu 86 5 Ser 54 6 Lys 80 7 Val 71 8 Leu 82 9 Trp 51 i0 Phe 64 ii Pro 48 12 Arg 21 13 Lys 37 14 Val 32 15 Ser 17 16 Glu 122 17 Leu 34 18 Asp 114 19 Lys 12

Repetitive Yield = 91%

A N-Terminal Sequence of Trypsin-Digested Tyrosine Hydroxyiase

158 BOVINE: (--X--) gly glu set lys val leu phe arg lys val set g ~ leu asp lys

157 RAT:. arg se t ala arg glu asp lys val pro phe arg lys val set glu leu asp lys (Grima et aL, 1983)

ser 158-~ [arg454 tTH ser40

ser 19., ] ser 153 TH ser 8,.] | | "] i ~arq157

Figure 2A: N-terminal sequence of bovine adrenal trypsin-digested tyrosine hydroxylase (tTH) compared with the native enzyme. The bovine protein sequence is shown in comparison with the rat sequence deduced from a cDNA clone by Grima et al. (4). The N- terminus of bovine tTH corresponds to postion 158 of the rat holoenzyme; an arginine at 157 was presumably the N-terminal site of trypsin digestion. (-) indicates that a predominant amino acid was not identified.

Figure 2B: Schematic representation of the predicted structure of tTH compared with native tyrosine hydroxylase (TH). The N- terminus of tTH corresponds to amino acid 158 with respect to the holoenzyme as determined by amino acid sequencing (see Figure 2A). A C-terminal trypsin cleavage in the vicinity of arginine 454 was predicted as explained in the text. tTH does not contain the four phosphorylation sites in the N-terminal region of the enzyme (serine residues 8, 19, 40, and 153) identified by C~p--~6~Tl et al. (8).

1 4 5 0


The predicted structure of tTH with respect to the native

enzyme is illustrated schematically in Figure 2B. As indicated,

this catalytic region occurs in the central and C-terminal

portion of the enzyme; the N-terminus of tTH corresponds to

postion 158. Since the resultant fragment is 39 kDa, there is a

second trypsin cleavage site approximately 45 amino acids (5 kDa)

from the C-terminus. Examination of the sequence of the rat

enzyme reveals an arginine at position 454; cleavage at this site

would generate a fragment of the correct molecular weight.

However, C-terminal analysis of tTH is required to define the

exact C-terminal trypsin digestion site. As is apparent in Figure

2B, the sequence of TH corresponding to the trypsin fragment does

not include the N-terminal phosphorylation sites (8). Consistent

with the predicted model (2,4), the regulatory region of TH is

distinct from the catalytic domain.

The sequence of the catalytic region of TH corresponds to

the region of the enzyme which shares 70% homology with PAH (5,6)

and TPH (7). This is in contrast to the N-terminus which shares

no significant homology and to the C-terminus which is 34%

homologous with PAH (6). These data indicate that the catalytic

regions of these hydroxylases are selectively conserved. The

sequences of the catalytic regions of PAH or TPH have not been

determined. However, Iwaki et al. (17) have reported that the

catalytically active 35kDa region of PAH is generated by

proteolysis of an ii kDa fragment from the N-terminus and a 5 kDa

fragment from the C-terminus of the native enzyme. This 35 kDa

catalytic region of PAH aligns with the sequence of native TH

which contains the 34 kDa tTH and is highly homologous to PAH.

To determine the characteristics of the catalytic domain of

TH compared with the native enzyme,the kinetic parameters of the

tTH and native TH were assessed. Assays were conducted at pH 5.8

for native TH and at pH 6.1 for tTH since, in agreement with a

previous report (2), we found that proteolysis resulted in a

shift in pH optima~ As determined by Lineweaver-Burk kinetic

analysis (Table II) bovine tTH has a lower K m for both substrate

(1.6 x 10-5 M) and cofactor (8.0 x 10 -5 M) than the native enzyme

(3.9 x 10 .-5 M and 2.9 x 10 -4 M~ respectively)~; the apparent Vmax

in the presence of either substrate or cofactor was appropriately

increased for tTH" Similar results have been reported for

proteolytic activation of rat TH (3). These data indicate that

pro~teolysis ~ctivates~ TH ~ia an~ incTease i~ ~ffinity for both


Table II: Kinetic constants of bovine trypsin-digested TH compared with the native enzyme. Bovine TH was purified as described (ii) up to the DEAE-cellulose column. The enzyme was incubated in the presence or absence of trypsin (0.5 mg/ml) for 15 min at 30°C in i0 mM potassium phosphate buffer pH 7.0. Enzyme activity was determined by the method of Coyle (12) as modified by Reis et al. (13) in the presence of 100 mM sodium acetate at pH 5.8 for TH or at pH 6.1 for tTH. Michaelis constants were determined by Lineweaver-Burk kinetic analysis . The K m for 6MPH 4 was assessed in the presence of i00 uM tyrosine; the K m for tyrosine was determined in the presence of 1 mM 6MPH 4.

Tyrosine 6MPH 4 Km Vmax Km Vmax (um) (nmols/7'/mg) (um) (nmols/7'/mg)

Native 39 +_ 5 10.3 +_ i.I 286 + 4 9.7 _+ 1.2

Trypsin-digested 16 ~ 2 21.5 ~ 1.0 80 +_ 7 22.3 + 13

substrate and cofactor. Although the trypsin fragment contains

the catalytic site, and thus the binding sites for substrate and

cofactor, the N-terminus of the native enzyme imposes additional

constraints for binding.

In summary, the catalytic domain of TH is contained within a

34 kDa region of the enzyme, the sequence of this region is

highly homologous to the corresponding regions of PAH and TPH.

Proteolysis results in removal of phosphorylation sites and

corresponds to an increase in affinity for both substrate and

cofactor. Further experiments are necessary to define the role of

the N-terminus in the regulation of cofactor binding and in

directing substrate specificity.

ACKNOWLEDGEMENTS

The authors acknowledge Onyou Hwang for many helpful discussions and for critical evaluation of the manuscript and Tony Fonzi for excellent technical assistance.

i.

2.

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1452


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characterization of the catalytic domain of bovine adrenal tyrosine hydroxylase

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