The N-terminal domain mutant binds to heparin and is phosphorylated by CAMP-dependent protein kinase .... dard assay conditions: 100 pM tyrosine, 500 pM 6-methyltetrahydro- pterin, 14 mM .... tion techniques free of such complications.
Protein Science (1993), 2, 1452-1460. Cambridge University Press. Printed in the USA. Copyright 0 1993 The Protein Society
Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase
S. COLETTE DAUBNER,' DANIEL L. LOHSE,'
' Department of Biochemistry and Biophysics and Department
AND
PAUL F. FITZPATRICK',2
of Chemistry, Texas A&M University,
College Station, Texas 77843-2128
(RECEIVEDApril 28, 1993; REVISEDMANUSCRIPT RECEIVEDJune 14, 1993)
Abstract Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophanhydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that theC-terminal two-thirds are homologous, while the N-terminal thirds are not.This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, twomutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutantbinds to heparin and is phosphorylated by CAMP-dependent protein kinase at thesame rate as the holoenzyme but lacks any catalytic activity. The C-terminaldomain mutantis fully active, with V,,, and K,,, values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal330amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, oneregulatory and one catalytic. Keywords: catecholamine biosynthesis; domains; kinetics; mutagenesis; phenylalanine hydroxylase; phosphorylation; tetrahydrobiopterin; tyrosine hydroxylase
Tyrosine hydroxylase catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine(L-DOPA), the first and the rate-limiting step in the pathway that yields the catecholamine neurotransmitters dopamine, norepinephrine, and epinephrine (Wiener, 1979; Zigmond et al., 1989). The enzyme is a tetramer of identical subunits of 498 amino acid residues (Grima et al., 1985). A metalloprotein, it contains one ferrous iron atomper subunit (Fitzpatrick,1989; Haavik et al., 1991). Tyrosine hydroxylase belongs to a group of enzymes that display marked functional and structural similarities. Other membersare phenylalanine hydroxylase and tryptophan hydroxylase (Kaufman & Fisher, 1974). Allthree contain iron, catalyze ring hydroxylation of aromatic amino acids, and utilize tetrahydrobiopterin (BH,) as the physiological reducing substrate. All three arerate-limiting catalysts for importantmetabolic pathways: tyrosine hydroxylase, catecholamine biosyntheReprint requests to: Paul F. Fitzpatrick, Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128.
sis; phenylalanine hydroxylase, phenylalanine catabolism (Shiman, 1985); and tryptophan hydroxylase, serotonin biosynthesis (Fujisawa et al., 1982). The overall sizes of the three proteins differ: tyrosine hydroxylase contains 498 amino acid residues; phenylalanine hydroxylase, 452; and tryptophan hydroxylase, 444 (Kwok et al., 1985; Ledley et al., 1985;Grenett et al.,1987).Figure 1 shows the aligned amino acid sequences of all three proteins. Atvaline 164 of tyrosine hydroxylase, valine 117 of phenylalanine hydroxylase, andvaline 105of tryptophan hydroxylase, rea gion of striking identity begins and continues through the carboxyl-termini. Indeed, the three enzymes are 60%identical in their 330 C-terminal amino acids. Furthermore, comparison of thesequences of the rat tyrosine hydroxylase and human phenylalanine hydroxylase genes shows each consisting of I3 exons, with 10of 12intronlexon junctions conserved; the divergence is at the 5' end of the gene (DiLella et al., 1986; Brown et al., 1987). In contrast to their structural similarities, the regulatory properties of phenylalanine hydroxylase and tyrosine hydroxylase are very different: phenylalanine hydroxylase
1452
1453
Domains of tyrosine hydroxylase is activated by preincubation with phenylalanine (Shiman & Gray, 1980), nothing similar occurs with tyrosine hydroxylase; phenylalanine hydroxylase is activated by lysolecithin (Abita et al., 1984), tyrosine hydroxylase is not; phenylalanine hydroxylase is activated by thiol modification (Parniak & Kaufman, 1981), tyrosine hydroxylase is inactivated (Petrack et al., 1968); tyrosine hydroxylase is activated by heparin (Stone, 1980), phenylalanine hydroxylase is not. Both enzymes are activated by phosphorylation, but the sites and even the numbers of phosphorylation sites differ (Shiman, 1985; Haycock, 1990); in both cases the phosphorylation sites are serines in the N-termini (Kwok et al., 1985; Haycock, 1990). The locations of the phosphorylation sites and the correlationbetween the divergent regulatory properties and the divergent N-terminal sequences suggest that the N-termini of these proteins are discrete regulatory domains (Campbell et al., 1986; Grenett et al., 1987). Conversely, the C-terminal twothirds of each protein would be catalytic domains, containing a common catalytic core. Consistent with such a model, limited proteolysis of phenylalanine hydroxylase removes an 11,000-Da polypeptide from the amino terminus (Iwaki et al., 1986). Mild trypsin treatment cleaves bovine tyrosine hydroxylase at amino acid 158 (Abate et al., 1988). Both of these sites are close to theproposed junction between domains.
In order to test the model of tyrosine hydroxylase as a tetramer of subunits that contain N-terminal regulatory domains and C-terminalcatalytic domains, mutant proteins were designed and expressed that consist only of the portions of the enzyme thought to comprise these domains. This paper describes the design, purification, and some properties of these proteins.
Results and discussion
Expression and purification of catalytic and regulatory domains The region of homology between the three tetrahydropterin-dependent hydroxylases begins at valine 164 of tyrosine hydroxylase (Fig. 1). Togenerate a protein containing only the putative regulatory domain, two consecutive stop codons were introduced into the sequence, replacing arginine 159 and aspartate 160. To generate a protein containing only the putative catalytic domain, the first 155 amino acids were deleted and a new start codon introduced. The constructs were designed to include as few artificial substitutions of amino acids as possible; however, due to thenecessity of placing a methionine at the N-terminus of the protein, theC-terminal domain included the mutation of valine 156 to methionine. The
80 MPTPSAPSPQ PKGFRRAVSE QDAKQAEAVTSPRFIGFtRQS LIEDARKERE AAAAAAAAAV ASSEPGNPLE AVVFEERDGN HMSTA VLENPGLGRK LSDFGQETSY IEDNCNQNGA 35 MIEDNKE NKDHSLERGR 17 160 AVLNLLFSLR GTKPSSLSRA VKWETFEAK IHHLETRPAQ RPLAGSPHLE YFVRFEVPSG DLAALLSSVR RVSDDVRSAR & S I E W V L R L EE&NDyNLTH -SRLKK D E Y W E T H U KRSLPALTNI IKILRHDIGA TVHELSRDKK 115 ATLIFSLKNE E L L I M L K I E Q E K H m L HIESBKSKRRN SEFEIEVDCP TNREQLNDIF HLLKSHTNVL SVTPPDNFTM97
M m R K VSELDKCHHL VTKFDPDLDL D H P W S D Q W R Q R R X L I U I AFQYKHGEPI PHVEYTJ4EEI T w m R T IQELDRFANQ ILSYGAELDA DllPWKDPW RARRXQFADI AYNYRHGQPI PRVLYMEEEK K E E G L E S w m K K ISDLDHCANR VLMYGSELDA DHPGIKDNVY RKRRXYFADL AMSYKYGDPI PKMFTEEEI
ED KD
230 185 171
ATMKEVYVTL KGLYATRACR LHLEGFQLLE RYCGYR~OSI PQLLDVSFWL KERWQLRP v u ; L L s m r LASXAERVFQ 310 KTWGTVFKTL KSLYKTRACY LYNHIFPLLE KYCGFHEDNI PQLUIVSQIL QTCTGFRLRP t0U;LLssmr LGGLAFRVFH 265 KTWGTVFREL NKLYPTRACR z Y L m L P L L s NDCGYRPDNI PQLEDISWL KERTGBSIRP VAGyLsPmr L S G ~ V F H 251 390 345 331 AGLLSSYGIL AGLLSSFGIL AGLLSSISXL
LHSLSEEPEV QYCLSEKPKL KHVLSGHAKV
RAFDPDTAAV QPYQDQTYQP VYFVSESIND AKDKLRNYAS RIQRPISVKF DPYTLAIDVL 470 LPLELEKTAI QNYTVTEFQP LrnrAISMD AKEWRNFAATIPRPISVRY DPYTQRIEVL 425 VYFVSISIED AKEIWREFKK TIKRPIGVKY NPYTRSIQIL 411 KPFDPKITYK QECLITTFQD
DSPHTIQRSL EGVQDELXTL AHALSAIS 498 DNTQQLKILA DSINSEIGIL CSALQKIK 453 KDAKSITNAM NELRHDLDW SDALGKVSRQ LSV
444
Fig. 1. Primary sequences of rat tyrosine hydroxylase (toh), human phenylalanine hydroxylase (pah), and rabbit tryptophan hydroxylase (trp). Residues that are identical among all three proteins are in bold type, whereas residues that are identical only between tryptophan and phenylalanine hydroxylase N-termini are underlined.
sh
S.C. Daubner et al.
1454
N-terminal domain contained no artificially introduced residues. Both the N-terminal and C-terminal domain mutants were expressed in Escherichia coli. Expressionof the N-terminal domain was several-fold higher than either the catalytic domainor the wild-type enzyme, 10,2, and 3% of the total protein,respectively. It proved possible to purify the N-terminal domainto homogeneity with the same protocol as is used for thewild-type enzyme. This involves ammonium sulfate precipitation followed by asingle heparin-Sepharose column (Daubner et al., 1992). The N-terminal domain bound to the heparin column,localizing the binding site for heparin to this region of tyrosine hydroxylase. Because it had no catalytic activity, the protein was monitored during purification by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A 1-L cell culture yielded approximately 8 mg of pure protein. Consistent with the binding of theN-terminal domain to the heparin column, the C-terminal mutant did not bind to heparin-Sepharose and was purified by a different procedure. A purification summary appears in Table 1; a DEAE column, ammonium sulfate fractionation, and a MonoQ columnyielded pure enzyme. A typical preparation was begun with 18 g cell paste. The overall yield in a preparation varied between 20 and 30%, with a typical final specific activity of 2 pmol/min-mg protein. Such a preparation could be carried outin 2 days. A photograph of an SDS-polyacrylamide gel containing representative samples of wild-type, C-terminal domain, and N-terminal domain tyrosine hydroxylase appears in Figure 2.
W Fig. 2. SDS-polyacrylamide gel electrophoresis of purified tyrosine hydroxylase domains. The samples are wild-type rat tyrosine hydroxylase(left), theC-terminal domainmutant(center),and the N-terminal domain mutant (right).
standard.An value of 14.2 was determinedfor the C-terminaldomain,andan value of 4.2 forthe N-terminus. These values yield a predicted value for the extinction coefficient of the intact protein of 10.0, in reasonable agreement with the reported value of 10.4 (Haavik et al., 1988; Daubner et al., 1992). For the C-terminal Spectral properties protein, the dye-binding assay gave a value that was 1.54Amino acid analyses were performed on both mutant pro- fold that obtained from aminoacid analysis, whereasfor teins, and theresults were compared to values from specthe N-terminal protein the Bradford assay value did not trophotometric measurements and from the dye-binding need correction. assay of Bradford (1967) using bovine serum albumin as Absorbance spectra for the two domains and for wildtype tyrosine hydroxylase appear in Figure 3. The spectrum of the C-terminal domain was similar to that of wild-type tyrosine hydroxylase on a molar basis, with the Table 1. Purification of the C-terminal same absorbance maximum,278 nm. The spectrum of the domain of tyrosine hydroxylase" N-terminal domain was quite different. Consistent with . the unusual aromatic amino acid content (eight phenylTotal alanines, one tyrosine, no tryptophan), the absorbance Total protein Specific Yield peak is broad and has discrete shoulders at 259 and 267 Step (mg) activity ('70) and a low extinction value. Lysate I65 1,250 0.13 100 Fluorescence emission spectra for the threeproteins apDEAE sepharose 89 22 I 0.40 54 pear in Figure 4. Whether excited at 260 or 280 nm, the (NH4)zSO.q 30-40'70 72 82 44 0.88 wild-type enzyme and the C-terminal domain emitted I6 20 2.04 33.7 Mono-Q maximally at 336 nm. The N-terminal protein showed peak emissionat 308 nm upon excitationat 260 or 280 nm, a From 20.5 g of cells. One unit will form I pmol of DOPA per min at 32 "C under stanagain consistent with the lack of tryptophan residues. The dard assay conditions: 1 0 0 pM tyrosine, 500 pM 6-methyltetrahydrosum of the emission spectra of the two domains was pterin, 14 mM P-mercaptoethanol, 10 pM ferrous ammonium sulfate, slightly less in magnitude than that of the intact protein 75 pg/mLcatalase,50 mM 2-(A"morpholino)ethanesulfonic acid, pH 6.5. and was blue-shifted about 5 nm, suggesting a change in ~
~~~~~~~
~
~
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~~
~~~
~~
~
~
~~~
~~
~
~~
~
~~
~
~
~~~
~
~~
~
~~
~
1455
Domains of tyrosine hydroxylase
r
20
60
I
50 40
I
30 20
X
10
0
Y
-10
0
240
280 320 360 wavelength, nm
185 225
400
Wavelength (nm)
Fig. 3. Absorbance spectra of wild-type tyrosine hydroxylase (O),the C-terminal domain (0),and the N-terminal domain (+) at pH 7 .
the environment of tryptophanylresidues upon removal of the N-terminal domain (results not shown). The CD spectra of the two mutants and of wild-type tyrosine hydroxylase appear in Figure 5 . Both the nearand far-UV spectra of the two domains are distinctly different. The lack of near-UV absorbance for the N-terminal domain is consistent with the lack of tryptophanyl residues. The decreased ellipticity in this region for the C-terminal domain compared to the wild-type protein
suggests that the environment Of at least One Of the three tryptophanyl residues has changed somewhat in the mutant. This is consistent with the fluorescence changes noted above. The strongsignal at 210 nm for theC-terminal domain is consistent with a relatively high helical content, while the spectrum for theN-terminal domain is consistent with a higher content of &sheet for the latter polypeptide. Simulations of the spectra by the method of Yang et al. (1986) confirmed this conclusion (results not
?-
z -80 250
270
290
310
Wavelength (nm) Fig. 5 . Circulardichroicspectra for wild-typetyrosinehydroxylase ("--), the C.terminal domain (- - -), and the N-terminal domain (- - -1 at p~ 7. A: F ~ ~ - uB:v Near-UV. .
shown). Below 210 nm, the sum of the spectra of the isolated domains agreed well with that of the native protein, consistent with a lack ofsignificantstructuralchanges in the isolated domains (results not shown).
Native tyrosine hydroxylase is a tetramer of identical subunits. The molecular weight of the C-terminal domain was determined gel by filtration, yielding an M, value of
0.6
of
0.4
0 290
1
r
Molecular weight
0.8
0.2
245
205
contact
330 370 41 0 wavelength, nm
450
Fig. 4. Emission spectra for wild-type tyrosine hydroxylase (O), the C-terminaldomain (O),andtheN-terminaldomain (*) atpH 7, 15 "C. The excitation wavelength was 280 nm. Similar results were obtained with excitation at 260 nm.
161,000 (results not shown). Thisis consistent with a tetramer 39,000 Mfor , subunit this protein, truncated similar to the wild-type enzyme, and suggests that this portion of the polypeptide chain also contains anintersubunit region. Kinetics
There was no detectable catalytic activity samples in of the N-terminal mutant, consistent with this being a regulatory .~ domain' In contrast, the domain had Significant Catalytic activity. A number of steady-state kinetic parameters were determined for the C-terminal mu-
I456
S.C. Daubner et al.
tant of tyrosine hydroxylase. For all of the parameters examined, there was no significant difference between the C-terminal domain mutant protein and wild-type tyrosine hydroxylase (Table 2). These results clearly establish that no aminoacid residues required for catalysis are located in the N-terminal 155 amino acids of tyrosine hydroxylase. This is the result expected if this constitutes a regulatory domain, whereas the C-terminal 330 amino acids constitute the catalytic core. The spectral results discussed above suggest that there are subtle structural differences between the intact protein and the two domains. Both the near-UV CD and fluorescence spectra suggest there is a change in the environment of one or more tryptophanyl residues in the C-terminal domain. Obviously, this change is not large enough to affect the catalytic ability of this mutant.
Phosphorylation In the intact protein, the N-terminus is the site of phosphorylation by protein kinases. Consequently, phosphorylation of the separate domains by CAMP-dependent protein kinase was examined. Both mutantswere exposed to in vitro phosphorylating conditions as described previously for wild-type tyrosine hydroxylase (Daubner et al., 1992). The N-terminus could be phosphorylated stoichiometrically, as can the wild-type enzyme (Fig. 6A). The C-terminal domain was not phosphorylated even during incubations 40 times longer than used for wild-type enzyme. By using smaller amounts of protein kinase at 10 "C, phosphorylation rates could be compareddirectly; the N-terminal domain was phosphorylated at essentially the same rate as wild-type tyrosine hydroxylase (Fig. 6B).
Thus, phosphorylation by CAMP-dependent protein kinase of the N-terminal domain occurs to the same rate and extent as with intact tyrosine hydroxylase. Although the N-terminal domain lackscatalyticactivity,as expected, it therefore still behaves as a correctly foldedregulatory domain. In addition,this polypeptide retains the ability to bind heparin, localizing the heparin-binding ability of intact tyrosine hydroxylase to the aminoterminal one-third.
Conclusion
We present strong evidence that the amino-terminal onethird and the carboxyl-terminal two-thirds of tyrosine hydroxylase constitute discrete regulatory and catalytic domains. Critically, the K,, and V,,, values for the protein lacking the first 155 amino acids are identical to those of the intact enzyme. This result establishes clearly that no aminoacid residues required for catalysis are located in the first 155 amino acids. This also demonstrates that the C-terminal domain mutantis able to fold independently
1
0.4
w
Table 2. Steady-state kinetic parameters f o r the C-terminal domain of rat tyrosine hydroxylasea ~~
-
-~
-
~~
~
~~~
-~ -~ -- ~~
~~~~~
~
C-terminal domain
Wild-type enzyme ~
~~~
-
~~
~ ~
-~~ -
~ ~~~
~
~~
-
~
0.1 o.2
*
~-~~-~ ~~
-~
221 i 19.9 ? 2.7 20.6 3.5 25.2 f 10.6 8.1 ? 0.8 k 3.0
205 f 11.6 16.5 f6.7 3.0d 41 f 4.0d 40.8 t 5.6 8.6 f 2.gd f 4.8 19.9 16.0 ~
Q I
0
-~-~ ~-~ ~
B
1 1
1
1
2
time, minutes
~~~
~~
a Conditions: 14 mM 6-mercaptoethanol, 10 bM ferrous ammonium sulfate, 75 p g / p L catalase, 50 mM HEPES, pH 7.0, 30°C. Determined at 525 pM 6-methyltetrahydropterin, varied tyrosine. Determined at 35 pM tyrosine. Daubner et al. (1992). e Determined at 100 pM tyrosine. 1 Determined at 500 pM tetrahydrobiopterin. g Determined with a dihydropterin reductase-coupled assay: 125 pM tetrahydrobiopterin, 200 pM NADH, varied phenylalanine, 5 pM ferrous ammonium sulfate, 75 &g/mL catalase, 50 mM HEPES-tetraethylammonium hydroxide, pH 7.0, 30 "C.
'
1I
Fig. 6. Phosphorylation of tyrosine hydroxylase domains. A: Stoichiometry of labeling. Wild-type tyrosine hydroxylase(O),the N-terminal domain (A),or the C-terminal domain(m) were incubated with CAMPdependent protein kinase (60 pg/mL) at30 "C. Aliquots were removed and tested for bound 32P at the times indicated as described in the Materials and methods. B: Rate of labeling. A total of 2.9 pM wild-type tyrosine hydroxylase (0)or the N-terminal domain(A)were incubated with CAMP-dependent protein kinase (6 pg/mL) at 10 "C. Aliquots were removed and tested for bound 32P at the indicated times as described in the Materials and methods.
1457
Domains of tyrosine hydroxylase
were from Pharmacia. Restriction and DNA modification enzymes were purchased from New England Biolabs and Promega. DNA sequencing used Sequenase from USB.
into an essentially native structure. The N-terminal domain similarly retains its ability to be phosphorylated at the same rate as thewild-type enzyme, and its ability to bind heparin. This is consistent with the domains retaining their native structures even when expressed separately. Previous studies on the domain fragmentsof tyrosine hydroxylase generated by proteolysis have been complicated by the difficulty of obtaining discrete, purified species (Abate et al., 1988). In this paper we describe the generation of the putative domainsby DNA recombination techniques free of such complications. The availability of these smaller, intact polypeptides should provide valuable tools to probe the structure of this physiologically critical enzyme.
Spectra Absorbance spectrophotometry was performed on a Hewlett-Packard 8452A diode array spectrophotometer. CD measurements were made using a JASCO 5-600 spectropolarimeter. Fluorescence emission spectra were recorded using an SLM 8000 fluorometer.
Construction of vectors for the expression of tyrosine hydroxylase domains The various constructions are summarized in Figure 7. Plasmid pTH5 contains the cDNA for rat tyrosine hydroxylase inserted into theBamHI site of pTZ18R as described previously (Daubner et al., 1992). To construct a plasmid coding for the C-terminal domainof rat tyrosine hydroxylase, a unique NdeI site was introduced to allow translation to begin at amino acid 156 of the tyrosine hydroxylase cDNA. Plasmid pTH5was subjected to mutagenesis using the oligonucleotide 5’-cgg gtg tct gac CAT ATG cgc agt gcc-3’ to generate ptTH1 (Kunkel et al., 1987); possible mutants were detected by screening for the
Materials and methods
Materials Oligonucleotides were custom synthesized on anApplied Biosystems Model 380B DNA synthesizer. Radiochemicals were from either New England Nuclear or Amersham Corp. The 6-methyltetrahydropterin was synthesized as described previously (Fitzpatrick, 1988). The (6R)-tetrahydrobiopterin was purchased from Research Biochemicals, Inc. Plasmid pTZ18R and helper phage M13K07
pETNTERM 5424 bp
pETOHAI55 5976 bp
ligate into pET3b
NdellBamHI digest, agarose gel, band isolation, NdellBamHl fragment
sc
/ I \‘x Sp P B
NdellBamHl digest, agarose gel, band isolation, NdellBamHI fragment
SpPB
x
Fig. 7. Construction of plasmids expressing tyrosine hydroxylase domains. Plasmid pETOHl is a pET3b plasmid containing the rat tyrosine hydroxylase cDNA between the NdeI site (5’ end) and the BamHIsite (3’ end). For the N-terminal domain, pTH6 (see Materials and methods)was subjected to mutagenesis to introduce a BamHI site after two new stop codons to halt transcription at serine 158; thenewvectorwascalled pTH6NTERM. For the C-terminus, pTH5 (see Materials and methods) was subjectedto oligonucleotide-directed mutagenesis to introduce an NdeI site changing valine 156 to methionine; the new vector was called ptTH1. The new NdeIBamHI fragmentswere inserted into pET3b. The final plasmids are not shown in circle form; pETNTERM is pET3b with the coding sequence for amino acids 1-158 inserted between the#JfO promoter and Tq4 terminator, and pTYHAlO5 is pET3b with the coding sequence for amino acids 156-498 inserted between the$10 promoter and T#Jterminator. Abbreviations for restriction endonuclease sites are as follows: A, ApaI; B, BamHI; E, EcoRI; H, HindIII; N, Ndel; P , PstI; Sc, ScaI (in thep-lactamasegene);Sp, SphI; X, Xmnl. The Ndel and BamHIsites used for recombination are shown in bold type.
1458 new NdeI site. Plasmid pETOHl is vector pET3b (Studier et al., 1990)containing the cDNAfor tyrosine hydroxylase between the NdeI and BarnHIsites. Plasmids pETOHl and ptTH1 were digested with NdeI and BamHI;both the pET3b portion of pETOHl and the fragment from ptTH1, which codes for the3' end of the tyrosine hydroxylase cDNA were purified from agarose gels and exposed to T4 DNAligase. Possible recombinants were screened by restriction enzyme analysis for the new smaller NdeIBamHI fragment and theloss of the PstI site at nucleotide position 166. One positive clone was sequenced to confirm the correct construction, that of the cDNA for the C-terminal domain of tyrosine hydroxylase inserted between the NdeI and BarnHIsites of pET3b, and named pETOHA155. To construct a plasmid coding for the N-terminus of tyrosine hydroxylase, mutagenesis was performed on pTH6 (Daubner et al., 1992); this plasmid was derived from pTH5 but has a new NdeI site at the start codon for the tyrosinehydroxylase cDNA. Two stop codons and a BarnHI site were introduced into the cDNAso that translation would stop after alanine159. The oligonucleotide used was 5'-c gtg cgc agt gcc TGA TAG gac aag gtc ccc tgg atc cca aga aaagtg-3'. Resulting clones were screened for the new BarnHI site. One positive plasmid was sequenced to confirm the correct construction and called pTH6NTERM. Plasmids pETOH 1 and pTH6NTERM were digested with NdeI and BarnHI; both the pET3b portion of pETOHl and the fragment from pTHGNTERM, which contains the 5' end of the tyrosine hydroxylase cDNA, were purified from agarose gels and exposed to T4 DNA ligase. Possible recombinants were screened by restriction enzyme analysis for the new smaller NdeIBarnHI fragment and the loss of the unique ApaI site, which is at nucleotide position 1,671 in the tyrosine hydroxylase cDNA insert. One positive clone was sequenced for the correct construction, that of the cDNA for the N-terminal domain of tyrosine hydroxylase inserted between the NdeI and BarnHI sites of pET3b. It was named pETNTERM. Protein purification Rattyrosinehydroxylaseexpressed in E. coli strain BL21(DE3) pLysE was purified as described previously (Daubner et al., 1992). The catalytic subunit of bovine heart cyclic AMP-dependent protein kinasewas purified by the method of Flockhart and Corbin (1984). Catalase was obtained from Boehringer Mannheim. To obtain the N-terminal domain, competent cells of the E. coli strain BL21(DE3) pLysS transformed with pETNTERM were grown overnight at 37 "C in LuriaBertanimediumplus 100 pg/mLcarbenicillinand 50 pg/mL chloramphenicol. This was diluted 100-fold into 1 L of the same medium at 37 "C. Expression was induced by addition ofisopropyl P-D-thioglucanopyranoside
S.C. Daubner et ai. (IPTG) to 0.5 mM when an A600 valueof 0.7 was reached. After 2 h cells were collected by centrifugation at 9,000 X g for 30 min. Cell pellets were stored overnight at -20 "C. All further isolation steps were performed at 4 "C. The cell pellet (2.4 g) was suspended in 8 mL of 50 mM Tris-HCI, 100 pM EDTA, 1 pM leupeptin, 1 pM pepstatin, 100 pg/mLphenylmethylsulfonylfluoride (PMSF), pH 7.5. The cell suspension was sonicated using four bursts of 30 s at 45 W with 30-s intervals. The lysate was passed through an 18-gauge needle, then centrifuged at 27,000 X g for 60 min. The supernatant was brought to 0.01% polyethyleneimine by the addition of a 0.5% stock in 20 mM Tris-HCI, pH 7.0,stirred in the cold for 20 min, and then centrifuged at 27,000 x g for 20 min. The supernatant was treated with ammonium sulfate; protein precipitating between 20 and 30% saturation was collected and dissolved in 50 mM Tris-HC1, 10% glycerol, 0.1 mM EDTA, 1 pM leupeptin, 1 pM pepstatin, pH 7.0. This was applied to a 1 x 3-cm column of heparin-Sepharose preequilibrated with the same buffer. The column was washed with the same bufferuntil the Azsovalue of the effluentwas below 0.05. The N-terminal domain was eluted with a 100-mL gradient of the same buffer containing 0-0.7 M KCI. Samples were assayed by SDS-polyacrylamide gel electrophoresis(Laemmli, 1970); those exhibiting a single band with an apparent molecular weight of 18,000 were pooled and stored at -70 "C. To obtain the C-terminal domain, competent cells of E. colistrainBLZl(DE3)pLysStransformed with pETOHA155 were grown overnight at 37 "C in LuriaBertani medium plus 100pg/mL carbenicillin and 50 pg/mL chloramphenicol. This was diluted 100-fold into 4 L of the samemedium at 37 "C. Expression was induced by the addition of IPTG to 0.4 mMwhen an A600value of 0.5 was achieved. After 3h the cells were centrifuged at 9,000 x g for 10 min at 4 "C, and the pellets were stored overnight at -20 "C. The cell pellet (21.5 g) was suspended in 100 mLof cold 50 mM Tris-HCI, 100 pM EDTA, 1 pMleupeptin, I pMpepstatin, 100 pg/mL PMSF, pH 7.5, anddispersed by passage through a 13G syringe needle. The cell suspension was lysed by sonication with cooling using four bursts of 30 s at 45 W with 1-minrestintervals. The lysate was centrifugedat 27,000 x g for 60 min and the pellet discarded. The supernatantwas applied in four separate aliquots to an HR 10/30 column packed with 8.5 mL of DEAESepharose FastFlow in 50 mM Tris-HC1,O. 1 mM EDTA, 10% glycerol, pH 7.5. After loading the sample, the column was washed with the same buffer for 5 min at 2.0 mL/min. A 30-mL linear gradient of 0-0.675 M KC1 in 50 mM Tris-HC1,O.l mM EDTA, 10% glycerol, pH 7.5, was used to elute the enzyme. The fractions containing the greatest amounts of enzyme activity were pooled. Ammonium sulfate fractionation was performed; protein precipitating between 35 and 45% saturationwas collected. The pellet was dissolved in 30 mL of 50 mM Tris-HC1,
1459
Domains of tyrosine hydroxylase 0.1 mM EDTA, 10% glycerol, pH 7.5. Five-milliliter aliquots of this were applied to a MonoQ HR 5 / 5 column equilibrated with 50 mM Tris-HC1,O.1 mM EDTA, 10% glycerol, pH 7.5. After loading, the column was washed with the same buffer for4 min at 0.5 ml/min. A 30-mL linear gradient from 0 to 0.85 M KC1 in 50 mM Tris-HC1, 0.1 mM EDTA, 10% glycerol, pH 7.5, was used to elute the enzyme. The fractionswere assayed for tyrosine hydroxylase activity; those with the highest specific activity were pooled.
and Ritchie (1991) was used. The conditionswere as follows: CAMP-dependent protein kinase (catalytic subunit), [ Y ~ ~ P I A T(0.1 P mM, 130,000 cpm/nmol), and MgC12 (10 mM), in 50 mM HEPES-tetraethylammonium hydroxide, pH 7.0. When determining stoichiometries of labeling, the protocol was carried out at 30°C with 60 pg/mL protein kinase. Rates of phosphorylation were determined at 10 "C with 6 pg/mL kinase.
Acknowledgments Gel filtration The molecularweight of the catalytic domain was determined using a Superose 12 HR 10/30 column equilibrated with 50 mM sodium phosphate, 150 mM NaCI, pH 7.5, at 4 "C. Each protein was injected and efuted at a flow rate of 0.5 mL/min, and the absorbance of the eluatewas monitored continuously at either 280 or 226 nm. The standards used were RNAse A (1 3,700), chymotrypsinogen (25,000), ovalbumin (46,000),bovine serum albumin (66,700), aldolase (158,000), catalase (232,000), and ferritin (440,000).
Assays The assay of tyrosine hydroxylase was based on the release of tritium from 3,5-[3H]tyrosine asdescribed previously (Fitzpatrick, 1989). Kineticparameters were determined as described previously (Fitzpatrick, 1991) in 50 mM MES, pH 7.0, at 3 0 T , unless indicated otherwise. Steady-state kinetic data were fit to the relevant programs of Cleland (1979), using the KinetAsyst software (IntelliKinetics, State College, Pennsylvania). Phenylalanine hydroxylase activity was measured by recording the oxidation of NADH at 340 nm using a coupled assay with dihydropterin reductase. The range of phenylalanine concentrations was 5-380 pM with 125 pM tetrahydrobiopterin, 50 rnM HEPES-tetraethylammonium hydroxide, 75 pg/mL catalase, 5 pM ferrous ammonium sulfate, 200 pM NADH, 0.45 unit/mL sheep dihydropterin reductase, pH 7.0, at 30°C. Several methods of determining the concentration of tyrosinehydroxylase were used.Whenassaying crude samples the dye-binding method of Bradford (1967) was used, with bovine serum albumin as the standard protein. When samples were pure or nearly pure protein estimation was made by Azso measurements. Both domains were subjected to amino acid analysis to determine the extinction coefficients. Amino acid analyses were performed by Dr. Mark Wright of the Texas A&M University Biotechnology Support Laboratory.
Phosphate labeling For incorporation of 32Pinto tyrosine hydroxylase by CAMP-dependent protein kinase, the method of Roskoski
This research was supported in part by NIH grant GM 47291, Robert A. Welch Foundation grantA-1245, American Heart Association grant-in-aid 9100890 to P.F.F., and American Heart Association (Texas Affiliate) grant 92R-033 toS.C.D. P.F.F. is an Established Investigatorof the American Heart Association. We thank Dr. Alain Chaffotte for the simulations of the CD spectra.
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