deletion mutagenesis of rat pc12 tyrosine hydroxylase regulatory and catalytic domains

Journal of Molecular Neuroscience Copyright �9 1993 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/93/4(2): 125-139/$3.80

Deletion Mutagenesis of Rat PC12 Tyrosine Hydroxylase Regulatory and Catalytic Domains

Paula Ribeiro, * Yuehua Wang Bruce A. Citron, and Seymour Kaufman

Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, MD 20892

Received September 21, 1992; Accepted November 2, 1992

Abstract

The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains. A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified from E. coil T h e results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity. On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme. These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455. Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparent K m for tyrosine and phenylalanine substrates, and a substantial increase in the K i for dopamine inhibition. The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition. The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor. These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.

Index Entries: Tyrosine hydroxylase; mutagenesis; dopamine regulation.

Introduction

Tyrosine hydroxylase (tyrosine 3-monooxygenase EC 1.14.16.2) catalyzes the first, rate-limit- ing step in the biosynthesis of catecholamine

neurotransmitters, including dopamine and nor- adrenaline. In the reaction, tyrosine is converted to 3,4-dihydroxyphenylalanine (Dopa) in the presence of molecular oxygen and the cofactor tetrahydrobiopterin (BH4) (Kaufman, 1974,1987).

*Author to whom all correspondence and reprint requests should be addressed.

Journal of Molecular Neuroscience 125 Volume 4, 1993

126 Ribeiro et al.

Tyrosine hydroxylase is inhibited by its substrate, tyrosine (Shiman et al., 1971; Ribeiro et al., 1991; Wang et al., 1991), and by its products, Dopa and the catecholamines (Nagatsu et al., 1964a; Kaufman and Fisher, 1974; Okuno and Fujisawa, 1985). In contrast, phosphorylation by cAMP- dependent protein kinase activates tyrosine hydroxylase (Kaufman, 1987), causing a decrease in the K m for the cofactor, a shift in the pH optimum, an increase in the K i for catechol inhibition, and an increase in Vma x at neutral pH. Other acti- vating factors indude high salt concentrations, polyanions, phospholipids, and limited proteolysis (Kaufman and Kaufman, 1985).

Early studies of the effects of proteolysis of tyrosine hydroxylase with trypsin (Petrack et al., 1968; Kuczenski, 1973) or chymotrypsin (Shiman et al., 1971) showed that the hydroxylase could be hydrolyzed to a catalytically active species with an apparent mol wt of 34 kDa (Musacchio et al., 1971; Hoeldtke and Kaufman, 1977; Vigny and Henry, 1981). The finding that the 34 kDa proteolytic fragment could no longer be activated by phosphorylation (Hoeldtke and Kaufman, 1977) indicated that this form of the enzyme con- tained the catalytic domain, but that the regulatory domain had been removed by the action of the protease. Recently, an analysis of the structure of the 34 kDa fragment of tyrosine hydroxylase showed that limited trypsin proteolysis removed a 17 kDa N-terminal fragment and a smaller 5 kDa C-terminal fragment from the 56 kDa monomer t (Abate et al., 1988; Abate and Joh, 1991). These studies also confirmed that the resulting 34 kDa species was catalytically active and lacked all four phosphorylation sites located in the N-terminus (Abate and Joh, 1991). The proteolyzed hydroxylase was further characterized as having many of the properties of the activated native enzyme, including a lower K m for BH 4 and a higher pH optimum. These properties are analogous to those of the phosphorylated

%he 56 kDa value is based on the primary structure of tyrosine hydroxylase (Grima et al., 1985), whereas Abate and coworkers (1988) used a value of 60 kDa based on the results of SDS-polyacrylamide gel electrophoresis.

native enzyme (Kauhnan, 1987), and appear to be caused by the removal of the phosphorylation sites (Abate and Joh, 1991). In contrast to the effect of phosphorylation, however, proteolysis also decreased the K m for the substrate tyrosine and broadened substrate specificity. These changes were not caused specifically by the removal of the phosphorylation sites, but were associated with the overall disruption of the N-terminus (Abate and Joh, 1991). These findings are consistent with the model that the N-terminus of tyrosine hydroxylase, like that of related aromatic amino acid hydroxylases (Kaufman, 1985), is the regulatory domain that directs cofactor binding and substrate specificity.

The present study has employed mutagenesis to further investigate the functional organization of tyrosine hydroxylase. Toward this goal, we have engineered a series of deletion mutations that selectively removed different size fragments from the regulatory and catalytic regions of the recombinant enzyme. The constructs were then expressed i n E. coli and purified for subsequent characterization. Depending on the size and loca- tion of the deletion, the mutant enzyme forms displayed different activities and distinct kinetic properties. Furthermore, in comparison to the wild-type, the mutants defined a region in the N-terminus that restricts substrate binding but facilitates the binding of catecholamine products and subsequent inhibition of the enzyme. These findings provide new insight into the regional organization of tyrosine hydroxylase and clarify the role of the N-terminus in the regulation of the enzyme.

Materials and Methods

Reagents GeneAmp polymerase chain reaction sys-

tems were obtained from Perkin Elmer Cetus (Norwalk, CT). DNA sequencing reagents (sequenase version 2.0) were obtained from United States Biochemical Corp. (Cleveland, OH).

Journal of Molecular Neuroscience Volume 4. 1993

Tyrosine Hydroxylase Domains 127

Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Boston, MA) and Bethesda Research Labs (Gaithersburg, MD) and used according to the manufacturers' recommendations. L-phenylalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine (Dopa), NADH, dihydropteridine reductase, ATP, and cAMP- dependent protein kinase catalytic subunit were obtained from Sigma (St. Louis, MO). 3-Hydroxy- tyramine (dopamine) was purchased from Calbiochem (La JoUa, CA). Rabbit antityrosine hydroxylase polyclonal antibody was purchased from Accurate Chemical and Scientific Corpora- tion (Westbury, NY). Horseradish peroxidase- conjugated goat antirabbit IgG was obtained from Bio-Rad (Richmond, CA). Catalase and protease inhibitors were from Boehringer Mannheim (Indianapolis, IN). (6R)-5,6,7,8-Tetrahydrobiop- terin (BH4) and 6-methyltetrahydropterin (6MPH4) were provided by D. Schirks Laboratories (Jona, Switzerland). Labeled amino adds, b(3,5-3H)tyro - sine (54 Ci/mmol) and L-(U-14C)phenylalanine (513 Ci/mol) were from Amersham (Arlington Heights, IL), and (0~-32p)-dATP was from New England Nuclear (Boston, MA).

Construction and Expression of Rat PC12 Tyrosine Hydroxylase cDNA Mutants The full length rat PC12 tyrosine hydroxylase

cDNA, cloned in plasmid pGEM3Z (Wang et al., 1991) was used as a template for polymerase chain amplification. The seven oligonucleotide primers used in the PCR reactions are summar- ized in Table 1. The polymerase chain reaction was used to introduce an NdeI site, which contains an initiation codon at the 5' end of the coding sequence. The amplification was carried out by 15 cycles of denaturation, annealing, and polymerization at 94~ for I min, 52~ for 2 min, and 72~ for 3 min with an automatic thermo- cycler. All of the fragments generated by PCR were passed through Chromaspin-400 columns (Clontech, Palo Alto, CA), digested with NdeI and BamHI, and then ligated into NdeI-BamHI digested pET3C expression vector (Studier et al.,

1990). The ligation mixtures were transformed to E. coli strain DH5 using standard procedures (Sambrook et al., 1989). The transformants were screened, the positive mutant clones were puff- fled, and their DNA sequences were verified by the dideoxy chain termination sequence method (Sanger et al., 1977).

All mutant clones confirmed by sequencing were retransformed to host strain BL21(DE3) for expression, unless otherwise indicated. Cell growth conditions and the preparation of crude extracts were as described previously (Wang et al., 1991). The wild-type and those mutants that displayed tyrosine hydroxylase activity were selected for further purification.

Partial Purification of Wild-Type and Mutant Forms of Tyrosine Hydroxylase Wild-type tyrosine hydroxylase can be purified

to homogeneity by a rapid procedure that involves ammonium sulfate fractionation followed by chromatography on a Heparin-Sepharose col- umn (Fitzpatrick et al., 1990; Wang et al., 1991). The mutants, however, did not bind to Heparin- Sepharose; therefore, alternative purification strategies were needed. In the case of the N-terminal mutants AA and AB (Fig. 1), between 30 and 40% of allmeasurable hydroxylase activity occurred in the form of insoluble inclusion bodies, not unlike that seen with the wild-type (Wang et al., 1991), and presumably the result of overexpres- sion in E. coli. Thus, the two mutants and the wild-type were purified directly from the inclusion bodies by using the method of Lin and Cheng (1991), with some modifications. Briefly, 1.5 g of mutant or wild-type E. coli cells were lysed by a combination of treatments that first removed the outer cell wall under hypotonic conditions and then disrupted the remaining sphero- plasts with mild sonication (Lin and Cheng, 1991). The inclusion bodies were pelleted by centrifugation and washed repeatedly in low levels (1%) of Triton X-100 to remove contaminating E. coli proteins (Lin and Cheng, 1991). The purified inclusion body pellet was subsequently resus-

Journal of Molecular Neuroscience Volume 4, 1993

128 Ribeiro et al,

Table 1 Summary of Oligonucleotides

Used in the Polymerase Chain Reaction

Primers Residues 5' N-terminal

Ol13 GGAAAGACAT ATGCCCACCCCCAGC 1 O 1 4 9 GGAAAGACAT ATGCGCTrCG AGGTGCCCAG T 133 O 1 5 7 GGAAAGACAT ATGGACCCTG ATCTGGACCT G 185 O 1 5 8 GGAAAGACAT ATGAGTGCCA GAGAGGACAA G 158

3' C-terminal O 1 5 0 GGAAAGAGGA TCCTrAGCTA ATGGCACTCA GTG 498 O 1 5 1 GGAAAGAGGA TCCTFAGAAT GGGCGCTGGA TACGA 455

Plasmid Mutant

pYHW5 WT

pYHW12

pYHW16 &B

pYHW14 &C

pYHWl 1 AD

pYHW13 &E

pYHW17 AF

pYHW15 AG

1 100 200 3O0

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIllllllllllllllllllllllllllllll~'//;,i i

i i

!

i i i

i

133 I i i

lso I 155

400 relative

498 aa MW activity

;'/~'/////,~ 56.0 kDa 1.0 |

i

41.7 kDa 1.5

I 39.0 kDe 2.5

35.7 kDa 0

' 455 51.2 kDa 0

I 37.0 kDa 0 i

I 34.2 kDa 0.5 i i

31.0 kDa 0

Fig. 1. Schematic representation of tyrosine hydroxylase deletion mutants. The seven mutant products (M-G) and the wild-type (WT) are shown. The regulatory domain is indicated by vertical lines and the horizontal lines depict the region that is predominantly catalytic. The tyrosine hydroxylase activities were measured in crude extracts and are relative to that of the wild-type (0.07 ~tmol/min/mg of protein).

pended in 5 mL of sonication buffer (50 mM Tris-HC1, 0.25M sucrose, 0.1 mM EDTA, 2 wV/ leupeptin, 0.5.WV/pepstatin, 100 ~tg/mL phenyl- methylsulfonyl fluoride [PMSF], pH 7.2) and subjected to 15 pulses of sonication (50 W), each for 30 s, followed by a 30 s pause. At this stage, sonication was found to be the most effective method for the disruption of the protein aggregates in the inclusion bodies and the consequent solubiliza- tion of active tyrosine hydroxylase. Other solu- bilizing procedures tested, including the use of various detergents and denaturation/renatur- ation techniques (Frankel et al., 1991; Lin and

Cheng, 1991; Wang et al., 1991), all resulted in complete loss of enzyme activity. After sonication, soluble wild-type and mutant tyrosine hydroxylase were recovered by centrifugation at 17,800g for 15 min. Each enzyme form was then precipitated by an ammonium sulfate fractionation between 30 and 42% of saturation, and the resulting pellets were resuspended in a minimum volume of 20 mM Tris-HC1, 0.1 mM EDTA, 10% glycerol, 2 WV/leupeptin, 0.5 p_M pepstatin, pH 7.2, to a final protein concentration of 1-2 mg/mL.

In contrast to AA and AB, the mutant lacking both 157 amino acids from the N-terminus and



43 amino acids from the C-terminus (Fig. 1) was expressed predominantly as a soluble protein, with <5% of total measurable activity being associated with the insoluble inclusion body pellet. Consequently, this mutant was purified directly from the soluble cytosolic fraction, after disruption of E. coli cells. Approximately I g of cells were resuspended in the same sonication buffer as above and were subjected to 15 pulses of sonication (50 W), each for 30 s, followed by a 30 s pause. The lysates were centrifuged at 17,800g for 15 min and the soluble enzyme was precipi- ta ted by an a m m o n i u m sulfate fractionation between 30 and 42% of saturation. The resulting pellet was resuspended in 50 mM MES, 2 W~I leupeptin, 0.5 WVI pepstatin, 100 ~tg/mL PMSF, pH 7.0, and 500-~tL aliquots containing approx 1.5 mg of protein were added to 143 ~tL of a calcium phosphate gel suspension (30 mg/ mL). The samples were shaken for 20 min at 4~ and the gel was washed once in the same buffer. The enzyme was subsequently eluted from the gel by resuspension in 125 ~tL of 0.125M potassium phosphate buffer, pH 7.4. The samples were shaken for another 10 rain and centrifuged. The supernatant containing the mutant hydroxylase was saved and the gel discarded.

Electrophoresis and Western Blot Immunoanalysis Standard SDS/polyacrylamine gel electro-

phoresis and Western transfer were performed according to the specifications of NOVEX (San Diego, CA), the manufacturer of the equipment. Immunoanalysis was carried out with mouse antityrosine hydroxylase polyclonal antibody (1:250 dilution), as otherwise described (Towbin et al., 1979; Wang et al., 1991). Densitometry of gels was performed with the IMAGE software package (O'Neil et al., 1989), and a peripheral COHU model 4815 solid-state video camera. Par- tially purified mutants were quantitated by densitometry, with the use of pure tyrosine hydroxylase as a standard. Typically, five differ-

ent concentrations of the standard, ranging from 0.5--4.4 ~tg were loaded with each electrophoretic separation. The resulting standard curve was consistently linear (r 2 > 0.99) in this range.

Assay Procedures The hydroxylation of tyrosine was measured

in 200 mM sodium acetate, pH 6.0, by the triti- ated water-release assay (Nagatsu et al., 1964b), with the modifications described previously (Ribeiro et al., 1991). Phenylalanine hydroxylation was assayed in 200 mM sodium acetate, pH 6.0, or 50 mM potassium phosphate, pH 6.8, by the method of Katz et al. (1976), as described by Ribeiro et al. (1991). The reactions with each substrate were initiated by the addition of either BH 4 or 6MPH 4 in the indicated concentrations, and the incubations were continued for 5 rain at 37~ For determination of pH curves, the assays were carried out in 50 mM MES buffer or 50 mM HEPES buffer in a range of pH 5.5-8.5, as described previously (Wang et al., 1991). When the K i for catechol inhibition was to be determined, BH 4 was varied (15--250 ~M) at a fixed tyrosine concentration (20 WVI) with either Dopa or dopamine in a range of 0.5-250 ~M. The K i values were obtained from secondary plots of slope vs catechol concentration.

The binding of 3H-dopamine to partially purified wild-type and mutant tyrosine hydroxylase was assayed in 50 mM sodium phosphate, pH 7.0, containing 10 mM MgCI2, 100,000 cpm 3H- dopamine with enough nonradioactive dopamine to produce the desired concentration, and 16-34 ~tg of wild-type or mutant tyrosine hydroxylase in a total volume of 100 ~tL. After a 5 min incubation at 30~ bound and free ligand were separated by gel filtration on Sephadex G-50, as described previously (Ribeiro et al., 1992). Bind- ing data were corrected for nonspecific binding and fitted to a one-site model by computer- assisted, nonlinear regression analysis (Munson and Rodbard, 1980). Nonspecific binding was determined experimentally in the presence of a 1000-fold excess of nonradioactive dopamine,


130 Ribeiro et al.

and fitted as an independent parameter in the model (Munson and Rodbard, 1980). The estimated level of nonspecific binding was approx 7% of total binding.

Other Methods Pure recombinant tyrosine hydroxylase was

obtained as described previously (Wang et al., 1991). Protein was measured by the method of Bradford (1976) with the Bio-Rad assay kit and bovine serum albumin as a standard. The concentrations of tetrahydrobiopterin solutions were determined from the absorbance at 266 nm in 0.1N HC1 with the use of an extinction coefficient of 16 mM -1 x cm -1 (Shiman et al., 1971). All enzyme kinetic parameters were determined by computer-assisted, nonlinear curve fitting to the Michaelis-Menten model.

Results

Survey of Expressed Mutant Clones A series of rat PC12 tyrosine hydroxylase

cDNA deletion mutants were amplified by PCR, which generated clean cDNA fragments of the predicted sizes. Each product was cloned into pET3C vector and the tyrosine hydroxylase protein was expressed in E. coU.

Seven mutants were constructed that con- tained different deletions of tyrosine hydroxylase domains, as shown schematically in Fig. 1. Crude extracts from the mutant clones were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting, and the sizes of the expressed proteins were as predicted (data not shown). Among the seven constructs, three were found to have measurable tyrosine hydroxylase activity in crude extracts (Fig. 1) and were selected for further purification and characterization. Muta- tions that deleted the N-terminal 132 amino acids (AA) and 157 amino acids (AB) led to an increase in activity relative to the wild-type (WT),

but further deletion of up to 184 N-terminal residues (AC) total ly des t royed the act ivi ty of the enzyme. Similarly, the removal of the carboxyl-termina143 amino acids generated enzyme species that were inactive (AD, AE, and AG). The single exception was the mutant AF, which lacked both the 43 carboxyl-terminal amino acids and 157 amino acids from the N-terminus. This mutant showed measurable tyrosine hydroxylase activity equal to about 50% that of the wild-type.

Partial Purification and Characterization of AA, AB, and AF

Figure 2 shows a typical SDS-polyacrylamide gel of partially purified wild-type and mutants. The identities of the bands were established by Western analysis with the use of a polyclonal anfityrosine hydroxylase antibody. The molecular weights were determined from the relative mobility of suitable protein standards.

In order to make meaningful comparisons between the specific activities of the mutants and that of the wild-type, it was necessary to estimate the amount of enzyme protein in each of the partially purified preparations. In this study, the amounts of wild-type and mutant proteins were estimated by densitometry and used for subsequent calculations of specific activity. Typically, the amount of protein in the band of interest was determined by comparison with known amounts of pure tyrosine hydroxylase, as described in the Methods section. Alternatively, the amount of enzyme was determined by equating the purity of the preparation with the relative density of the band of interest. The two methods gave approximately the same results.

Several kinetic properties of the mutant forms of tyrosine hydroxylase were investigated and compared to those of the wild-type. Figure 3 shows the activities of the various enzyme forms as a function of the concentration of the substrate, tyrosine, in the presence of 115 ~M BH 4. The



1 6 0 . 0 - -

8 0 . 0 - -

4 9 . 5 n

3 2 . 5 - 2 7 . 5 -

W'l" AA AB AF

Fig. 2. SDS-polyacrylamide gel electrophoresis of partially purified wild-type (WT) and mutant (&A, AB, and AF) tyrosine hydroxylase. The bands of interest were identified by Western transfer and immunoblotting, as described in the Methods section. The densitometric scans illustrate the relative purity of the different preparations.

I000"

500 �84

0 0 I ~ 2~ 3~

Tyrosine, ttM

Fig. 3. Comparison of the activities of wild-type (WT), and the three mutant forms of tyrosine hydroxylase (AA, AB, and AF) as a function of tyrosine. Aliquots of wild- type, AA, AB (0.2-0.3 I.tg), and AF (0.8-1 ~tg) were assayed at pH 6.0 and at a constant BH 4 concentration of 115 pdgl. The reactions were carried out for 5 rain at 37~ as described in the Methods section. The activity is expressed in n m o l / m i n / m g of enzyme.

results illustrate how increasing deletions of 132 amino acids (AA) and 157 amino acids (AB) from the N-terminus of tyrosine hydroxylase led to a corresponding increase in catalytic activity. Con- versely, the deletion of a small fragment in the carboxyl terminus from a mutant already lacking in a large section of the N-terminus (AF) caused a substantial decline in activity, to less than 50% of the wild-type level. In the presence of BH 4, the three mutants and the wild-type all shared the same typical substrate inhibition at concentrations above 50 gM. Substrate inhibition was not evident, however, when the widely used cofactor analog 6MPH 4 was substituted for BH4 (data not shown).

Table 2 summarizes several kinetic properties of partially purified wild-type enzyme and mutant tyrosine hydroxylase forms. All three mutants and the wild-type enzyme were able to catalyze the hydroxylation of phenylalanine, in addition to tyrosine. At pH 6.0, and in the presence of BH 4, the mutants hA and AB showed consistently higher activities than the wild-type, both with phenylalanine and tyrosine; the apparent Vma x values for each of these substrates were between


132 Ribeiro et al.

Table 2 Summary of Various Kinetic Properties of Wild-Type

and Mutant Forms of Tyrosine Hydroxylase

Parameter WT AA AB AF

K m tyr (6MPH4) a WVI 48 27 27 49

Vmax U / m g 0.80 0.92 1.5 0.32

S0. 5 tyr (BH4) b ~VI 10 4.4 5.5 20

appVma x U/mg 0.53 0.65 1.20 0.21

K m phe (6MPH4) c W~I 160 85 82 120

Vmax U / m g 0.08 0.09 0. 19 0.06

K m phe (BH4) d W~I 62 32 27 60

Vma x U / m g 0.59 0.65 0.91 0.23

K m BH4(tyr) e WVI 56 49 59 43

Vmax U / m g 0.43 0.55 1.1 0.17

Ki/dopamine ~ 3.7 nd 41 8.6

K D dopamine pA4 1.5 nd 8 nd

K i Dopa WVI 63 nd 77 100

pH optimum 6.8-7.2 nd 6.8-7.2 nd

Wild-type (WT) tyrosine hydroxylase and the mutant forms AA, AB, and AF were assayed for tyrosine or phenylalanine hydroxylation, as described in the Methods section. The units of hydroxylase activity (U) are ~tmol of substrate hydroxylated per min of reaction per mg of enzyme.The amount of enzyme protein per reaction tube typically varied between 0.2 and 0.3 ~tg for the wild-type, AA and AB, and 0.8-1.2 p.g for AF. For determinations of kinetic parameters with phenylalanine in the presence of 6MPH 4, the amount of each enzyme species was increased to 1-3 p~g/tube.The concentration of enzyme protein in each partially purified preparation was determined by densitometry, as described in the Methods section.

aKm value for tyrosine was obtained by varying tyrosine between 5 and 200 ~ at a fixed concentration of 1 mM 6MPH4;

bS0. 5 represents the concentration of substrate that elicits half-maximal velocity at a

fixed BH 4 concentration of 115 W~I, and appVm~ is the apparent maximum velocity. A proper K m value could not be obtained because of substrate inhibition in the presence of BH4;

r concentration of 6MPH 4 was kept constant at 1 mM and that of phenylalanine was varied between 15 and 505 pM;

aThe concentration of BH 4 was fixed at 115 Wgl and that of phenylalanine varied between 10 and 105 ~,I.

eThe concentration of tyrosine was 20 ~VI and that of BH 4 varied between 15 and 500 W~d;

flnt-dbition constants (K i) and binding constants (KD) were measured as described in the Methods section, (nd), not determined.



1.1- and 2.3-fold higher, and the apparent K m (S0.5) values about twofold lower, than those of the wild-type. On the other hand, AF had consistently lower Vmax values with tyrosine and phenylalanine, and the apparent K m values were equal to or higher than those of the wild-type. When 6MPH 4 was substituted for the natural cofactor, there was a slight increase in the Vmax as a function of tyrosine with each of the enzyme species, and their respective K m values were all increased approx fivefold (Table 2). In contrast, the K m values for phenylalanine in the presence of 6MPH 4 were typically threefold higher and the Vmax values about tenfold lower than those measured with BH 4 (Table 2). Similar results were obtained when the reactions with phenylalanine were carried out at pH 6.8 (data not shown). These findings suggest that in the presence of 6MPH 4 there was a greater decrease in the activity with phenylalanine than with tyrosine, but the relative differences between the wild-type and the mutants were essential ly the same as those observed with BH 4. Finally, the mutants and the wil d- t yp e were found to have the same pH optima, which ranged from approx 6.8-7.2, and the same K m for BH 4, although a substantial change in both these parameters was expected following the disruption of the N-terminus (Abate and Joh, 1991).

Among the kinetic parameters measured in this study, the most marked alteration produced by a deletion of N-terminal residues was the K i for dopamine inhibition, which was increased approx tenfold when a single deletion removed 157 amino acids from the N-terminus (AB). On the other hand, the additional removal of a 43 amino acid segment from the carboxyl end (AF) seemed to reverse this effect on the K i for dopamine, so that its value was comparable to that of the wild-type (Table 2).

T h e K i for Dopa inhibition of the wild-type was nearly 20-fold higher than that for dopamine and, in contrast to dopamine, the K i for Dopa did not appear to respond to any of the deletions tested (Table 2). Other differences between Dopa and dopamine in the inhibition of tyrosine hydroxy-

lase are apparent in Fig. 4. In competition studies with BH 4, the inhibition of wild-type tyrosine hydroxylase (WT) by dopamine decreased as the concentration of BH 4 increased. Although the inhibition pattern was not that of a classical com- petitive inhibitor, the overall effect of dopamine was to increase the K m for BH4, with a minimal change in apparent Vmax- Essentially, the oppo- site effect was observed when Dopa competed with increasing concentrations of BH 4. The K m for BH 4 did not change significantly, but the Vma x was greatly decreased in response to Dopa, in a man- ner consistent with classical noncompetitive inhibition. Similar patterns of inhibition by Dopa and dopamine were observed with the two mutants AB (Fig. 4) and AF (data not shown).

The specifc binding of 3H-dopamine to partially purified wild-type and AB was characterized as having apparent binding constants (KD)

of 1.5 and 8 gM, respectively (Table 2). The estimated binding capacities were 2 and 9 pmol/~tg enzyme for the wild-type and mutant, respectively. The results suggest that the decreased sensitivity of AB to dopamine inhibition is caused by a decrease in its affinity for the catecholamine.

Discussion

Partial proteolytic digestion of native tyrosine hydroxylase showed that the enzyme is com- posed of two main functional regions, a catalytic domain and a regulatory domain, which is involved in phosphorylation, cofactor binding, and substrate specificity (Hoeldtke and Kaufman, 1977; Abate et al., 1988; Abate and Joh, 1991). From these earlier studies it was also known that the regulatory domain was disrupted by the proteolytic removal of an N-terminal 17 kDa fragment, and that the catalytic domain did not include this N-terminal fragment or a C-terminal 5 kDa region (Abate and Joh, 1991). The present investigation of several tyrosine hydroxylase mutants has expanded on these earlier findings, and has further defined the localization of


134 Ribeiro et aL

0.02

0.01

~ -0.04

dopamine

! . . . . . . . I

0.00 0.04 0.08

0.04-

Dopa 0"06-1WT . /

t / 250pM

0.04] / ~,,,,10011M

/ ,r / 50 pM

~ 0 ~IM �9 �9 - - m u m

-0.04 0.00 0.04 0.08

!

-0.04

dopamine 0.03] ~ Dopa

/ 2 5 0 pM ] / 2 5 0 tiM /

0.02] /

~ 1 0 0 pM 1 ~ I00 IIM

5o .M o . o , ] d . ~ . . ~ ~ 2, ,M 25 pM

0.00 0.04 0.08 -0.04 0.00 0.04 0.08

1 / [ BH 4 ] Fig. 4. Product inhibition of partially purified wild-type tyrosine hydroxylase (WT) and mutant AB by dopamine and

Dopa. The concentration of BH 4 was varied (15-250 pM) at each of the indicated concentrations of inhibitor. The substrate tyrosine was kept constant at 20 p.M. 1/V is the reciprocal of the rate, measured in nmol/min/mg of enzyme; 1/[BH4] is the reciprocal of the concentration of cofactor in gM.

the catalytic domain and the role of the N-terminus in the regulation of the activity of the enzyme.

The present results show that mutations that deleted up to 157 N-terminal amino acids generally caused an increase in catalytic activity, but further deletion of up to 184 N-terminal residues totally destroyed the activity of the enzyme. These results confirm that the catalytic domain does not include the N-terminal 17 kDa region (Abate et al., 1988; Abate and Joh, 1991) and further indicate that the left-end of the catalytic domain must be located between residues 158 and 184. On the carboxyl side, the results show that the removal of the C-terminal 43 amino acids

caused a complete loss of activity, unless this C-terminal deletion was accompanied by a deletion of 158 residues from the N-terminus, as in mutant AF. The results make it unlikely, therefore, that this 43 amino acid deletion has entered the right most edge of the catalytic domain, which would be expected to consistently destroy catalytic activity. Instead, this region may be involved in the folding of the catalytic site, or perhaps, as suggested recently (Liu and Vrana, 1991), in main- taining the tetrameric conformation of the holoenzyme. The finding that the two deletion construct AF has lower activity than the wild-type enzyme is surprising because this is the mutant



that most closely resembles the proteolytically activated 34 kDa species (Abate et al., 1988; Abate and Joh, 1991). Although there may be several explanations for this apparent difference, the most probable is that our deletion mutation and the proteolytic cleavage occurred on different residues, and thus may not be directly comparable.

Previous studies of proteolyzed adrenal tyrosine hydroxylase revealed that the N-terminus was responsible for directing substrate binding, such that the progressive digestion of N-terminal residues caused a proportional increase in activity and the affinity for the substrate (Abate and Joh, 1991). The present study gives additional credence to these earlier findings. The evidence reported here shows that the hydroxylase activities with tyrosine and phenylalanine were stimu- lated by the removal of 132 N-terminal amino acids, and were increased even further when the deletion was lengthened to 157 amino acids. Fur- thermore, the mutations caused an approx 50% decrease in the apparent K m (S0.5) for both tyrosine and phenylalanine. The present results thus are in accordance with the model proposed by Abate and Joh (1991) that a region in the N-terminus serves to restrict substrate binding, presumably by preventing access of the substrate to the active site.

In contrast to the earlier model, however, the present results do not support the notion that the N-terminus increases substrate specificity (Abate and Joh, 1991). Previous studies of proteolyzed tyrosine hydroxylase reported that the removal of the N-terminus caused a preferential decrease in the K m for phenylalanine and tryptophan (Abate and Joh, 1991), suggesting that the N-terminus imposed stringent substrate binding requirements as well as an overall restriction on binding. This was not observed in the present study, in which the K m values for tyrosine and phenylalanine were similarly decreased by about 50% following the removal of up to 157 N-terminal residues. Although the precise reason for this difference is unknown, it should be pointed out that the wild-type recombinant hydroxylase has a lower K m for phenylalanine that the native

enzyme (Ribeiro et al., 1991; Wang et al., 1991), suggesting that the hydroxylase expressed in E. coli has less stringent substrate binding requirements than its mammalian counterpart. This may help to explain, in part, the apparent inconsistency between our results and those obtained with proteolysis of either bovine or brain tyrosine hydroxylase. It also raises the interesting possi- bility that some, as yet unknown eukaryotic posttranslational modification, which is missing in E. coli, may be required to impose substrate specificity. One such modification is suggested by recent evidence that the native enzyme contains small amounts of tightly bound catecholamines, which are coordinated to iron in the enzyme, and whose role is to maintain the hydroxylase in a state of low activity (Haavik et al., 1988). The effect of this posttranslational modification on the binding of amino acid substrates has not been clarified and deserves further investigation.

The region at the left end of the regulatory domain contains the four phosphorylation sites of tyrosine hydroxylase, including a serine resi- due at position 40 that is the substrate for cAMP- dependent protein kinase (Joh et al., 1978; Vulliet et al., 1980; Campbell et al., 1986; Haycock and Haycock, 1991). With native enzyme, the phosphorylation of ser 4~ decreases the K m for the pterin cofactor between three and tenfold and shifts the pH curve from an optimum of about 6.0 to a more physiological pH 7.0-7.5 (Kauhnan, 1987). The same changes in the K m for 6MPH 4 and pH optimum are caused by proteolytir digestion of the N-terminal region that contains all four phosphorylation sites (Abate et al., 1988; Abate and Joh, 1991), suggesting that the role of the N-terminus is inhibitory and imposes a constraint on the binding of BH 4 (Weiner et al., 1978). In contrast to the native enzyme, the recombinant tyrosine hydroxylase expressed in E. coli is purified in an activated form that has the same low K m for the cofactor and the same high pH optimum as either the phosphorylated or the proteolyzed native enzyme. The recombinant hydroxylase can be phosphorylated, but it cannot be further activated by phosphorylation (Wang et al., 1991). Accord-


136 Ribeiro et al.

ingly, the results in the present study indicate that the deletion of up to 157 amino acid residues from the N-terminus, which removes all the phosphorylation sites, does not affect either the K m for BH 4 or the pH optimum. One kinetic parameter, however, was dramatically altered by mutations that disrupted the N-terminus. The K i for dopamine inhibition was increased nearly tenfold when 157 amino acid residues were deleted from the N- terminus of the recombinant enzyme. The K i of the wild-type was virtually identical to that of native mammalian tyrosine hydroxylase, and the increase seen with the N-terminus mutant was comparable to that caused by phosphorylation of the native enzyme (Kaufman, 1987). In addition, the mutation was shown to decrease the affinity for dopamine binding, as evidenced by a fivefold increase in the K D value of the mutant relative to the wild-type. Thus, although the binding site(s) for dopamine do not appear to be located in the deleted N-terminus, this region appears to play a role in facilitating the binding of the catecholamine and the consequent down-regulation of the enzyme. Inexplicably, when the carboxyl end 43 amino acids were also deleted, as in the case of the mutant AF, the K i for dopamine was considerably lower than that of AB, and only twofold higher than that of the wild-type. The sig- nificance of this change is unclear, but it suggests that the right most edge of the C-terminal may serve to relieve the enzyme from some of the inhibitory functions of the regulatory domain.

These findings join a growing body of evidence that points to catecholamine inhibition as a eukaryotic posttranslational modification whose absence in E. coli may explain the activation of the cloned enzyme. An earlier study showed that the low levels of catecholamines that are tightly bound to the purified hydroxylase (Haavik et al., 1988) are rapidly dissociated under phosphory- lating conditions (Haavik et al., 1990). More recently, two separate investigations also showed that the high-affinity binding of dopamine to recombinant tyrosine hydroxylase reverses the kinetic activation of the enzyme, and permits reactivation by phosphorylation (Daubner et al.,

1992; Ribeiro et al., 1992). Thus, the loss of bound catecholamines may be the first response to phosphorylation that leads to subsequent kinetic activation. This would explain why a mutation in the N-terminus of the recombinant enzyme would cause a dramatic change in dopamine binding and inhibition, but would not, in the absence of dopamine, affect either the K m for the cofactor or the pH optimum.

The present results suggest that dopamine and the immediate product of tyrosine hydroxylation, Dopa, inhibited the enzyme by different mechanisms and with different potencies. In contrast to dopamine, Dopa did not increase the K m for BH 4 significantly, and behaved essentially as a noncompetitive inhibitor. The/~. for Dopa was nearly tenfold higher than that for dopamine and none of the mutations tested appeared to change that K i substantially. These differences between Dopa and dopamine inhibition of tyrosine hydroxylase have been reported previously (Kaufman and Fisher, 1974), and suggest that Dopa does not play the same crucial role as the catecholamines in the regulation of the enzyme.

In summary, in the present study we have characterized several deletion mutants of tyrosine hydroxylase whose activities and properties have helped to define the regional organization of the enzyme. For the most part, the results reported here support the model that has been proposed for tyrosine hydroxylase (Weiner et al., 1978; Abate and Joh, 1991), which in turn is comparable to that described for the related monooxygenase, phenylalanine hydroxylase (Iwaki et al., 1986). Figure 5 shows, in a schematic form, how the present data expanded on that earlier model. The results described here support the notion that tyrosine hydroxylase is organized into a regulatory domain located within the first 157 residues of the N-terminus, and a C-terminal catalytic region, which resides between residues 158 and 184 on the amino end, and up to position 455 on the carboxyl end. As suggested previously, the first 157 amino acids in the N-terminus of tyrosine hydroxylase have an overall inhibitory effect on the enzyme, such that the removal of


Tyrosine Hydroxylase Domains 13 7

-157 aalfrom

N'~ter:o0.

NH3 +

increase in activity decrease In dopamine affinity

- 4 3 aa from . ._

C - t e r m . " ~

-43 aa from ,.~ C-term.

COO"

Inactive

-157 aaJfrom N;erm.

COO"

NH3 +

Some activity and restored affinity for dopamlne

Fig. 5. Scheme illustrating the functional organization of the tyrosine hydroxylase monomer. The details of the model are discussed in the text.

this region causes an increase in activity and a decrease in the K m for the substrate. In addition, the model shows the iron center of tyrosine hydroxylase, which is thought to be the site for the binding of dopamine and other catecholamines (Andersson et al., 1988; Haavik et al., 1988). Removal of the inhibitory N-terminal 157 amino acid region dramatically decreased the affinity for dopamine and the sensitivity to dopamine inhibition, suggesting that an important role of this N-terminal region is to facilitate the binding of catecholamines and the subsequent down-regulation of the hydroxylase. Nei- ther the K m for the pterin cofactor nor the pH optimum were affected by the removal of these 157 residues, when significant changes in both these parameters would have been predicted (Abate and Joh, 1991). These results are consistent with earlier suggestions that these kinetic changes associated with the activation of tyrosine

hydroxylase are the indirect result of a decrease in the binding of catecholamines (Daubner et al., 1992; Ribeiro et al., 1992). Finally, the present data indicate that a small 43 amino acid region in the carboxyl end, which is located outside the catalytic domain, may serve to relieve the inhibitory role of the N-terminus. In combination with a deletion of 157 residues from the N-terminus, the removal of this C-terminal fragment restored the affinity for dopamine and generally reversed the kinetic changes caused by the disruption of the N-terminus. In the absence of other deletions, however, or in conjunction with less disruptive N-terminal mutations, the removal of the 43 amino acid fragment led to complete loss of activity, which emphasizes the importance of this region for main- taining the conformation of the active site. Col- lectively, these findings provide important new insight into the functional organization and the mechanisms of regulation of tyrosine hydroxylase.


138 Ribeiro et al.

Acknowledgments

The authors wish to thank M. Iwaki and J. P. Tipper for their expert advice and Cindy Falke for technical support.

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deletion mutagenesis of rat pc12 tyrosine hydroxylase regulatory and catalytic domains

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