The Effect of Ligands of Phenylalanine 4-Monooxygenase on the CAMP-dependent Phosphorylation of the Enzyme”

The rate of phospho~lation of phenylalanine 4-mon- ooxygenase by the CAMP-dependent protein kinase was found to be under substrate-directed regulation. Thus, t-phenylalanine made the hydroxylase a bet- ter substrate for the kinase, whereas the cofactor 1- ~~y~~~o-~,6,7,8-tetrahydrobiopterin (BHd was a negative effector. The deph~phorylation of the enzyme by the kinase in the presence of high concentrations of MgADP was also stimulated by phenylalanine and in- hibited by BH,. A kinetic analysis indicated that the effects of phen- ylalanine and BH4 were mediated by distinct sites cou-pled by a free energy of 3.2 kJ. mol”. Among the ligands tested, only phenylalanine and BH, affected the phosphorylation of the hydroxylase at physiologically relevant concentrations. Whereas higher Concentrations of several naturally occurring or synthetic amino acids acted like phenylalanine, the widely used synthetic cofactor 6,?-dimethyltetrahy-dropterin did not mimic the effect of BH4. Less phenylalanine was required to activate the phosphorylated hydroxylase (0.9 mol of phosphate/ subunit) than the dephosphorylated enzyme (0.07 mol of phosphate/subunit). This was true whether matrix of hepatocytes. The subcellular distri- bution of BH, is but it seems reasonable to assume a distribution in the cytosol matrix and nuclear matrix. Mo~hometric analysis (33) indicates that 45% of the liver volume is occupied by hepatocyte cytosol matrix and 53% by hepatocyte cytosol and nuclear matrix. A rough approximation of the concentration of the hydroxylase and BH, in the compartments be 1.1 mg/ 5.4 ppt'iiter, respectively. the M, of the subunit of the hydroxylase to be 51,000 (1, 15) and of BH, to be 241, the concentration of the hydroxylase of BH, the hydroxylase is be the main intracellular binder of BH, one site the cofactor of BH. theo- retically p~ depending on the degree of saturation of the hy~oxylase to cofactor. by spotting aliquots (40 pl) on phosph~ellulose strips (21). Similar results were obtained when the degree of dephosphorylation was assayed by the amount of "P becoming soluble in 10% trichloroacetic acid. leads to the doubly liganded PB-state. If the enzyme is first exposed to BH4 (B-state) and then to phenylalanine, a doubly liganded state (BPI occurs which differs from the PB-state in its p h o s p h o ~ l a t a ~ ~ t y . For the sake of simplicity, only one subunit of the oligomeric enzyme is considered

ably by binding to an allosteric site which is distinct from the substrate-binding site (5, 6). The hydroxylase is also a substrate for CAMP-dependent phosphorylation in vitro (7,8) and in vivo (9, lo), and the stimulation of the hydroxylase activity by this covalent modi~cation (7,11) seems at least in part to be due to sensitization of the enzyme towards activation by phenylalanine (12,13, this study).
The primary aim of the present study was to learn whether ligands of the hydroxylase, like phenylalanine, could modulate the CAMP-dependent phosphorylation of the enzyme. It was of particular interest to know if such ligand-modulated phosphorylation was likely to occur under physiologically relevant conditions, and if it might enhance the fine control of the hydroxylase activity by phenylalanine. A second aim was to characterize the specificity of the sites mediating the ligand effects as well as any interactions between such sites. Finally, the effect of phenylalanine and BH, on the kinase-catalyzed dephosphorylation of the phosphoenzyme was studied.

6,7-Dimethyltetrahydropterin was from Aldrich (Steinheim, FGR).
Tetrahydrobiopterin was generously donated by Hoffman-La-Roche (Basel, Switzerland). The compound was dissolved in 5 mM HCl and kept at -20 "C under N*. The particular batch used contained 70% of the R-diastereoisomer and 30% of the S-diastereoisomer, as analyzed' by high-performance liquid chromatography according to Bailey and Ayling (14). The concentrations of the cofactor given refer to that of the R-diastereoisomer.
The 3H-labeled phenylalanine was further purified by higb-performance liquid chromatography using a 50 X 0.3 cm column packed with Zipax SCX (du Pont Chemical Co.) and 10 mM sodium acetate (pH 3.9) containing 2% (v/v) ethanol as the mobile phase. The 3Hlabeled phenylalanine (tR = 3 min at a flow rate of 2 ml/min) was in this way separated from a radioactive contaminant having the same retention time as tyrosine (tR = 0.9 min).
Phenylalanine 4 -m o n~x y g e n~ was prepared from rat liver by a slight modification (15) of the procedure of Shiman et aE. (16). The preparation of enzyme used contained 0.07 mol of phosphate per subunit of M, = 51,000 and had a specific activity of 1300 nmolmin" . mg" with 6,7-dimethyltetrahydropterin as the cofactor. The phosphate content was assayed essentially as described by Stull and Buss (17). The concentrations of the hydroxylase given refer to the amount of enzyme subunits.  (21). Preparation of Phosphorylated Phenylalanine 4-Mo~waxygenuse-10 JLM hydroxylase was incubated for 30 min at 30 "C under phosphorylation conditions (see above). The concentration of C subunit was 50 nM and of ATP 60 p M (20 pCi/ml). Controls (nonphosphorylated hydroxylase) were obtained by incubation in the absence of C subunit. The preparation was desalted by passage through a column of Sephadex G-25 (Pharmacia, Uppsala, Sweden) equilibrated with Hepes buffer.
Assay of P~~y l a l a n~~ 4 -M o~~g e~e Activity-An aliquot (12.5 p l ) of hydroxylase, preincubated as described in the legend to Fig. 6, was added to 112.5 pl of assay mixture of give final concentrations of 15 mM Hepes-NaOH (pH 7.0), 2 mM EDTA, 3 mM dithiothreitol, 75 p~ BH,, 1 mM ['Hlphenylalanine (5 pCi/mll, and 1600 units/ml of catalase. Following an incubation for 2 min at 25 "C, the reaction was terminated by adding 25 p1 of 30% (w/v) aqueous trichloroacetic acid, and pr~ipitated protein removed by centrifugation (12,000 X g., for 4 min). Portions (80 pl) of the supernatant were injected into the liquid chromatograph using a 25 X 0.46 cm column packed with Spherisorb ODS (10 pm). The mobile phase (0.1 M NH40H adjusted to pH 4.6 with acetic acid and supplemented with 0.3 mM tetrabutylammonium hydrogen sulfate) was pumped at a flow rate of 1.5 mllmin. The fluorescence (excitation at 274 nm, emission at 304 nm) of the eluate was monitored with a Kontron spectrofluorimeter SFM 23. Tyrosine ( t~ = 2.6 min) has an approximately 100fold higher fluorescent intensity than phenylalanine (tR = 3.3 rnin). More than 95% of the applied tyrosine was recovered in the fraction retained between 2.5 and 3 min. In samples lacking enzyme (blanks) only 0.02% of the applied radioactivity was eluted in the fraction corresponding to the t~ value of tyrosine. The present assay differs from that of Bailey and Ayling (22) by using the ion-pair-forming tetrabutylammonium ion in the mobile phase. This modification shifted the order of elution of phenylalanine and tyrosine (see above), and considerably lowered the blank values.
It should be noted that by measuring the hydroxylase activity as the appearance of isotopically labeled tyrosine, interference from any unlabeled tyrosine carried over from the p~incubations was avoided. This was especially important for those experiments (to be shown in Fig. 6) where the hydroxylase was preincubated with both phenylalanine and BHI. For samples of hydroxylase not containing preformed tyrosine, identical values of enzyme activity were obtained whether formed tyrosine was detected by its fluorescence or its radioactivity. Protein was measured by the method of Bradford (23) using bovine serum albumin as the standard.

RESULTS
Phenylalanine and BH, Alter the Rate, but Not the Extent, of the Phosphorylation of Phenylalanine 4Monooxygenme-The rate of phosphorylation of phenylalanine hydroxylase by c A~P -d e~n d e n t protein kinase increased in the presence of phenylalanine and decreased in the presence of the natural cofactor BH, (Fig. 1). The magnitude of the responses to these effectors were independent of the source of the kinase used (CAMP-dependent protein kinase holoenzyme type I or the C subunit of type I1 kinase), The initial rate of phospho~lation was linear with respect to the concentration of kinase (range tested 0.5-100 nM) and time (Fig. 2). The onset of the effect of phenylalanine was rapid as compared to the time scale of the experiments (Fig.   2).
The apparent end point of 32P incorporation into the hydroxylase was not affected by substrate or cofactor ( Fig. 1  phosphorylation of the hydroxylase increased up to 2-fold when assayed in the presence of several analogs of phenylalanine3 (Table I). The synthetic analogs P-2-thienylalanine and p-chlorophenyialanine were moderately less potent, whereas the naturally occurring amino acids tested were at least 2 orders of magnitude less potent than phenylalanine. For all the analogs, the transition from no effect to full effect on the phosphorylation occurred in a relatively narrow concentration range ( Table I and   phenylalanine 4-monooxygenase The initial rate of phosphorylation of phenylalanine 4-monooxygenase (2 p~ with respect to subunits) was determined by analyzing duplicate aliquots removed after 4, 8, and 12 min of incubation for the amount of 32P incorporated. The incubations contained 100 mM (rather than 15 mM) Hepes-NaOH (pH 7.0), 60 p~ [y3*P]ATP, and 2 nM C subunit. The concentrations of effector given refer to the final concentrations in ihe incubations. The relative phosphorylation rate is the ratio between the rate in the presence and absence of effector (mean of three experiments, the range being less than *15% of the values shown). Parallel incubations were run in which kemptide or histone replaced hydroxylase as the substrate and the concentration of kinase was 0.2 nM. Except for a 20% inhibition by 20 mM tryptophane, none of the compounds tested significantly affected the rate of phosphorylation of kemptide or histone. The value in parentheses has been corrected for the assumedly nonspecific inhibition of phosphorylation by that amino acid as determined using kemptide as the substrate. decrease the rate of phosphorylation (Table I), in contrast to the natural cofactor BH, (Fig. 1, Table I).
The Kinetics of the Effects of BH, and Phenylalanine on the Rate of Phosphorylation of Phenylalanine 4-Morwoxyge1me-The phosphorylation of the hydroxylase decreased to about half its initial rate when the concentration of BH4 was increased ( Fig. 3A, 0). A Hill plot of the BH4 concentration dependence of the effect indicated a Hill coefficient of about 1 (Fig. 3B). Phenylalanine, in a concentration-dependent manner, increased the rate of phosphorylation up to %fold (Fig. 4 A , 0) with an apparent positive cooperativity as evidenced by a Hill coefficient of about 2 (Fig. 4C).
Testing of combinations of phenylalanine and BH4 revealed that the two ligands did not act independently. Thus, higher concentrations of BH, were required for inhibition in the presence of phenylalanine (Fig. 3B), and phenylalanine was a less potent stimulator in the presence of BH4 (Fig. 4 0 The lower potency of one ligand in the presence of the other could, however, not be ascribed to competition for a common site. In that case the effect of one ligand should be completely overcome by increasing the concentration of the other one, which did not occur (Fig. 3A, @ Fig. 4A, A and 0). The simplest model explaining all the data is that the interaction of one ligand with its binding site on the hydroxylase decreases the affinity of a separate site for the other ligand.
The theory of such linked binding of ligands has been treated by Wyman (26) and Weber (27). The Gibbs free energy of coupling (27) between the binding of phenylalanine and BH4 can be calculated from either Equation 1 or 2.
where Ki(BH4) is the apparent inhibition constant for BH4 (2.3 p~) in the absence of phenylalanine, and Ki (BHJPhe) is that constant (8.3 p~) at saturating concentration of phenylalanine (Table 11). Either equation gave the same result, i.e. AG = 3.2 kJ.mol-' for the free energy of coupling for the ligand pair phenylalanine and BH,.
At concentrations (data for 3 p~ and 20 p~ BH4 are shown) bordering the probable physiological range of free BH, , ' a more than %fold stimulation of the phosphorylation The concentration of the hydroxylase has been estimated to be 0.5 mg/g of rat liver (30) and that of BH, to be 2.8-2.9 g/g of rat liver (31, 32). The available evidence suggests that the hydroxylase is rate could be elicited by phenylalanine (Fig. 4A, 0 and A).
This may be related to the fact that physiological concentrations of BH, are subsaturating. According to the data of Fig.   located in the cytosol matrix of hepatocytes. The subcellular distribution of BH, is not known, but it seems reasonable to assume a distribution in the cytosol matrix and nuclear matrix. Mo~hometric analysis (33) indicates that 45% of the liver volume is occupied by hepatocyte cytosol matrix and 53% by hepatocyte cytosol and nuclear matrix. A rough approximation of the concentration of the hydroxylase and BH, in the relevant compartments would then be 1.1 mg/ liter and 5.4 ppt'iiter, respectively. Taking the M, of the subunit of the hydroxylase to be 51,000 (1, 15) and that of BH, to be 241, the concentration of the hydroxylase should be 21.8 p~ and that of BH,

p~.
If the hydroxylase is considered to be the main intracellular binder of BH, in hepatocytes and to have one binding site for the cofactor per subunit, the concentration of unbound BH. could theoretically vary between 0.6 and 22.4 p~ depending on the degree of saturation of the hy~oxylase with respect to cofactor. 3B, 3 p~ and 20 p~ BH, saturate 60 and 90%, respectively, of the BH4-binding sites in the absence of phenylalanine versus 25 and 70% at a high concentration of phenylalanine. This means that, due to the unfavorable energy of coupling between phenylalanine and BH4 just described, phenylalanine, in addition to its own direct positive effect on the phosphorylatability of the hydroxylase, can act indirectly by decreasing the binding of the negative effector BH4.
It was finally noted that hydroxylase which had been preincubated with phenylalanine and then exposed to BH4, was a better substrate than enzyme that had been preincubated with BH, and then exposed to phenylalanine ( Fig. 4B; see also Fig. 7).
The Effect of Ligands on the Reversal of the Kinase Reaction-The reversal of the protein kinase reaction (28,29) was studied by incubation of the 32P-labeled phosphohydroxylase with a high concentration of MgADP. In order to avoid interference from the forward reaction, the concentration of hydroxylase was kept low (0.1 FM). Furthermore, unlabeled ATP (20 /IM) was included to isotopically dilute the [y3*P] ATP formed in the reaction. Whether the ligands BH4 and phenylalanine were present or not, more than 95% of the 92P of the phosph~nzyme could be removed in a kinase/ADPdependent manner (Fig. 5, and data not shown).
The presence of BH, lowered the rate of dephosphorylation of phosphorylated enzyme (Fig. 5) , (A, A). Aliquots were tested for their hydroxylase activity as described under "Experimental Procedures." A , the activity of phosphorylated and nonphosphoryla~ enzyme as a function of preincubation conditions. The activity of enzyme preincubated in the absence of BH4 and phenylalanine was taken as unity. Half-maximal activation of the phosphorylated and nonphospho~lated forms of the hydroxylase required 29 and 51 ~L M phenylalanine, respectively. These values were the same whether the preincubation time was 5 or 10 min. When BH, was present, the corresponding values were 66 and 93 p~ for 5 min of preincubation and slightly higher for 10 min of preincubation. B, a Hill plot of the data obtained for phosphorylated (0) and nonphoshorylated (0) enzyme preincubated in the absence of BH4. Y = the fractional activation of the hydroxylase = v, -vJV,.,v,, where vx represents the activity of the enzyme preincubated with the concentration .
x of phenylalanine, u, represents the activity of the enzyme preincubated in the absence of phenylalanine, and Vmax represents the activity after preincubation with an optimal concentration of phenylalanine. The apparent Hill coefficient was 3 for the phosphorylated enzyme and 2.6 for the nonphosphorylated enzyme. shown (Fig. 6,O and A) for the activation of the hydroxylase activity of nonphosphorylated enzyme by phenylalanine were obtained under conditions similar to those used to test the stimulation by phenylalanine of the phosphorylation of the hydroxylase (Fig. 4 A , 0 and A).
Comparison of the two sets of data (Table 11) show a very similar dependence on the phenylalanine concentration for the two processes. Furthermore, the effect of phenylalanine revealed an apparent positive c~perativity in either case ( Fig.  4C and Fig. 6B).
The recent observation (12, 13) that the phosphorylated form of the hydroxylase required less phenylalanine to be activated was confirmed and shown to be true also when the physiological cofactor BH, was present (Fig. 6).

DISCUSSION
A large number of intracellular proteins are subject to phosphorylation by the catalytic subunit of CAMP-dependent protein kinases (34, 35). One way in which activation of this single type of kinase subunit may lead to various patterns of phosphorylation, depending on the metabolic state of the cell, is by metabolites interacting with substrate proteins whose susceptibility towards phosphorylation is thereby altered. The present study shows phenylalanine 4-monooxygenase to be under such substrate-directed control of phosphorylation, which has so far been described only for a few enzymes (36, 37), none of which are involved in amino acid metabolism.
Incubation of phenylalanine 4-monooxygenase with Lphenylalanine made the enzyme more readily phosphorylated by the C subunit of CAMP-dependent protein kinase (Figs. 1  and 2). Such modulation of the phosphorylation of the hydroxylase may also occur in uiuo, since phenylalanine acted at physiologically relevant (38) concentrations and was efficient in the presence of the natural cofactor BH, (Fig. 4). The hydroxylase is considered to have three types of sites capable of interacting with phenylalanine: a regulatory site responsible for the activation of the enzyme by phenylalanine (5, 6), the catalytic site, and a low-affinity site presumed to mediate enzyme inhibition by millimolar concentrations of phenylalanine (39). The strikingly similar dependence on phenylalanine concentration (Table 11) for the activation of the hydroxylase (Fig. 6) and the modulation of its rate of phosphorylation (Fig. 4), suggested that the same site, i.e. the regulatory site, mediated both effects of phenylalanine. This conclusion is further supported by the positive cooperativity shown by phenylalanine for both effects (Fig. 4, Fig. 6). Another clue as to whether the regulatory or the catalytic site was responsible for the enhanced phosphorylation came from the observation that in comparison with the nonphosphorylated hydroxylase, the phosphoenzyme required less phenylalanine to be activated (Fig. 6, Table 11), although it revealed the same I(, value for phenylalanine in the hydroxylation reaction (7; and data not shown). Based on simple considerations of thermodynamic reciprocity (27) it follows that the binding of phenylalanine to the regulatory site, but not to the catalytic site, should shift the equilibrium between nonphosphorylated and phosphorylated enzyme in favor of the latter. Such a shift occurs if phenylalanine preferentially enhances the forward reaction of Equation 3.
Hydroxylase + Mg[y-32P]ATP + "P. hydroxylase + MgADP (3) In fact, phenylalanine stimulated the forward reaction by a factor of 2 (Figs. 1, 2, and 4) as compared to 1.2-1.3 for the reverse reaction (Fig. 5). It follows that phenylalanine favored the phosphorylated form of the hydroxylase, in accordance with its action being exerted through the regulatory site. A preliminary "mapping" of this site (Table I) showed that it had a considerably (at least 500 times) higher affinity for the L-isomer than the D-isomer of phenylalanine. Whereas fl-2thienylalanine had a higher affinity than p-chlorophenylalanine for the regulatory site (Table I), the converse is true for the catalytic site (39). This means that the two sites differ considerably in the structure facing the phenyl moiety of phenylalanine. There has been some controversy as to whether tryptophan is an activator of the hydroxylase (5,6). The data of Table I support the contention (6) that this amino acid (at supraphysiological concentrations) does bind to the regulatory site.
Physiologically relevant4 concentrations of the natural COfactor BH4 decreased the rate of the CAMP-dependent phosphorylation ( Figs. 1 and 4) as well as the dephosphorylation of p~n y~l a n i~ 4 -m o~~g e~e The upper row gives the concentrations of phenylalanine required for half-maximal activation of the hydroxylase activity o f the nonphosphorylated and phosphorylated (values in parentheses) form of the enzyme. The values refer to enzyme preincubated in the absence of BH, or presence of 4 pM BH, and are calculated from Hill plots of the curves shown in Fig. 6A. The middle row gives the concentrations of phenylalanine half-maximally enhancing the rate of phosphorylation of the hydroxylase at various concentrations (0, 3, and 130 p~) of BH,. The values were calculated from the plots of Fig. 4, A and B. The lower row gives the concentrations of BH, half-maximally decreasing the rate of phosphorylation of the hydroxylase, as calculated from the experiments shown in Fig. 3 The values in parentheses refer to the phosphorylated form of the hydroxylase.
( Fig. 5) of the hydroxylase. A kinetic analysis (see under "Results") indicated that BH4 acted through a site distinct from the phenylalanine-binding sites on the hydroxylase. The concentration of BH, required to half-maximally modulate the rate of phosphorylation was 2.3 ~L M (Ki (BH4)) for the untreated hydroxylase and 8.3 p~ (Ki (BH,/Phe)) for the phenylalanine-treated enzyme (Fig. 3, Table 11). In comparison, the K, value for BH, is 2-3 p~ for nonactivated enzyme and 5-13 p~ for the enzyme activated by lysolecithin or by sulfhydryl modification (40,41). This concordance indicates that BH, modulates the phospho~lation by its binding to the active site of the hydroxylase. Furthermore, BH, obeyed Michaelian kinetics both as an effector of phosphorylation (Fig.  3) and as a cofactor in the hydroxylation reaction (9, 25,39-41).
Preincubation of the hydroxylase with a high concentration of BH, virtually blocks the activity effect (4, 6, 25), but not the phosphorylation-modulating effect (Fig. 4B) of phenylalanine. This means that the BH4-saturated hydroxylase remains in a low activity state (termed BP in Fig. 7) even when phenylalanine is bound to the regulatory site. Using the rate of phosphorylation of the hydroxylase as a probe, this doubly liganded state differs from the doubly ligandedphenylalanineactivated state (PB) as well as from the mono-liganded Pand B-states (see Fig. 7 for details). The latter (P, PB, and B) all differ from the ground state (G) in their phospho~latability (Fig. 7). Previous studies have provided ample evidence that the conformation of the P-state differs from that of the G-state (4, 16,40,42). A recent study using the susceptjbility t o chymotrypsinolysis as a conformational probe also concluded that the B-state of the hydroxylase differed from the G-state, but found no difference between states P and PB (42). This suggests that the binding of BH, to the native hydroxylase leads to a state (B) in which both the site(s) of c h y m o t~s i n cleavage and the site of phospho~lation have an altered microenvironment. When BH, binds to the phenylalanine-activated enzyme, the putative conformational change (from P to PB) may be more subtle, i.e. the change may be limited to the site of phosphorylation only. An alternative explanation, Le. that BH, interferes sterically with the phosphorylation, is unlikely because the synthetic cofactor 6,7-dimethyltetrahydropterin, which is a slightly bulkier molecule than BH, and binds to the same site, does not inhibit the phosphorylation reaction (Table I). Binding of phenylaIanine to the regulatory site of the hydroxylase under activating conditions (4) brings the enzyme to the P-state. Binding of BH, to enzyme in the P-state leads to the doubly liganded PB-state. If the enzyme is first exposed to BH4 (B-state) and then to phenylalanine, a doubly liganded state (BPI occurs which differs from the PB-state in its p h o s p h o~l a t a~~t y .
For the sake of simplicity, only one subunit of the oligomeric enzyme is considered in the model.

piF-rq
The magnitude of the phosphorylation enhancement by phenylalanine was 2-3-fold depending on the concentration of BH, present (Fig. 4). This may seem a modest effect, but as pointed out by Goldbeter and Koshland (43), there is an added sensitivity inherent in covalent modification schemes when one of the converter enzymes operates in the %eraorder" region, i.e. the region of saturation with respect to protein substrate. Phosphohydroxylase is dephosphorylated by a phosphatase ( I f ) , and the activity of this phosphatase may approach that predicted from zero-order (43) kinetics. Thus, the phenylalanine hydroxylase of rat liver hepatocytes, exposed to a brief stimulation of the CAMP-dependent protein kinase, remains in a phosphorylated condition after the activity of the CAMP-dependent kinase has returned to its basal level (44).
In conclusion, the data presented in this study indicate that phenylalanine 4-monooxygenase is subject to an interwoven control by allosteric binding of ligands and covalent modification by phosphorylation. Thus, an increase of the phenyl-