Proc. Nail. Acad. Sci. USA Vol. 89, pp. 10109-10113, November 1992 Biochemistry "7-Tetrahydrobiopterin," a naturally occurring analogue of tetrahydrobiopterin, is a for and a potential inhibitor of the aromatic hydroxylases (2-m ino-4-hydroxy-7-lydryropyl-(terytr)-S,6,7,8-terahydrpterinD MICHAEL D. DAVIS, PAULA RIBEIRO, JENNIFER TIPPER, AND SEYMOUR KAUFMAN Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, MD 20892 Contributed by Seymour Kaufman, July 24, 1992

ABSTRACT The ability of 2-amlno-4-hydroy-7-[diby- dependent of their respective substrates (7-9). droxylpropyl-(L-erythro)-5,6,7,8-tetrahydropterinJ ("7- In vitro, 4a-carbinolamine dehydrates at a rapid rate even in tetrahydroblopterin" or 7-Bil) to s e for the naual the absence ofthe dehydratase, but a small percentage ofthe cofactor tetrahydroblperin (BR4) has been studled in vitro In 4a-carbinolamine rearranges to form the 7-isomer (6, 7). Thus the U sof the three aromaic amin acd the dehydratase plays the dual role ofcatalyzing the dehydra- hydroxylases. With rat liver hydroxyse, the tion and preventing the isomerization ofthe cofactor(7). apparent K. for 7-BR4 is 160 FM, a value that Is 60-fold To date, the cause of the observed mild hyperphenylala- greater than that for the natural o a. In crast, the ninemia in children who excrete 7-BH4 remains unclear. hydroxylase rato is severely Inh by as lIe as 1 FM Among the possible reasons for this abnormality are either 7-BR4 when assayed In the preence ofphysiological concentra- that the reaction catalyzed by the dehydratase becomes tions of BR. This inhibition can be overcome either by an rate-limiting due to the absence of the or that 7-BH4 increase in the concea of BR4 or a decreae In the itself in some way impairs the phenylalanine hydroxylation cncentration ofphenyllanine. With both rat braln to reaction. One possible mechanism for such impairment be- hydroxylase and rat pCatyron hydoylae, came apparent when we showed that the oxidation of 7-BH4 the K.. value for 7-BR4 is about one order of greater by phenylalanine hydroxylase in the presence of phenylala- than the K. for B. Accordingly, 7-BR4 is a poor c tiive nine is 85-90%o uncoupled from the hydroxylation of the inhibitor of both t an ad tylroine hydroxyse. Thus, substrate (10). In this report we demonstrate an additional our results suggest that the oserved hyipe-heyalnnel in mechanism by which 7-BH4 might impair phenylalanine patients whoexcrete 7-BH4in their urine mayarisedirectly from hydroxylase activity; i.e., 7-BH4 is a potent inhibitor of this the inhibit of phenylalaine hydrolymby w levels ofthis reaction. Furthermore, we have examined the effect of pterin. On theother hand, it islessl y tatlow levels of7-BR4 7-BH4 on the reactions catalyzed by hydroxylase would affect the activity of i or try h hydrox e and hydroxylase and have found that although in viwo. these reactions are inhibited by this pterin in vitro, the inhibition is manifested only at very high concentrations of is an abnormality characteristic ofa 7-BH4. series ofdiseases that result from an inability to catalyze the first step in the catabolism of phenylalanine-namely, its MATERLS AND METHODS conversion to tyrosine (1). The most common cause of this abnormality is the absence of active phenylalanine hydrox- L-Phenylalanine, L-tyrosine, NADH, glucose 6-, ylase due to a defect in the gene coding for this enzyme (2, glucose-6phosphate dehydrogenase (from Leuconostoc me- 3). Variant forms of this disease have been reported that are senteroides), and m-hydroxylbenzylhydrazine were pur- caused by genetic defects in either the of the chased from Sigma. L-Tryptophan and beef liver catalase essential pterin cofactor tetrahydrobiopterin (BH4) or its were purchased from Calbiochem and Boehringer Mann- enzymatic regeneration (4). Since BH4 is required for cate- heim, respectively. Frozen rabbit brains, used in the prepa- cholamine and biosynthesis by tyrosine hydroxy- rationi of , were purchased from lase and tryptophan hydroxylase, respectively (4), patients Pel-Freez Biologicals. Sheep liver dihydropteridine reduc- with these other forms ofhyperphenylalaninemia also have a tase was purified through the calcium phosphate gel step (11). deficiency of biogenic amine . L-[3,5-3HJTyrosine (54 Ci/mmol; 1 Ci = 37 GBq) was from Recently, several patients have been described with a mild Amersham. 2-Amino-4-hydroxy-6-[dihydroxylpropyl-(L- form of hyperphenylalaninemia, who have elevated urinary erythro)-5,6,7,8-tetrahydropterin [(6R)-BH4 or tetrahydro- levels of an isomer of BH4, 2-amino-4-hydroxy-7-[1,2-dihy- ], 6-methyltetrahydropterin (6MPH4), and 7-BH4 droxypropyl)-(L-erythro)-5,6,7,8-tetrahydropteridine] ("7- were purchased from B. Schirks Laboratories, Jona, Swit- tetrahydrobiopterin" or 7-BH4),* and somewhat decreased zerland. All other reagents were of the highest quality avail- levels of BH4 (5). It has been suggested (6, 7) that the able. accumulation of abnormal amounts of 7-BH4 might be the Phenylaanine Hydroxylase. Phenylalanine hydroxylase result of an alteration of the gene coding for 4a-hydroxytet- was purified from rat liver by a combination of two methods rahydrobiopterin dehydratase (6, 7). This enzyme catalyzes the dehydration of 4a-hydroxytetrahydrobiopterin (4a- Abbreviations: BH4, tetrahydrobiopterin; 7-BH4, 2-amino-4-hy- carbinolamine), the form ofthe pterin that is initially released droxy-7-[dihydroxylpropyl-(L-erythro)-5,6,7,8-tetrahydropterin]; from the aromatic amino acid hydroxylases after the BH4- 6MPH4, 6-methyltetrahydropterin. *Due to the fluorescence properties of oxidized , urine samples are oxidized prior to analysis by HPLC. For this reason, The publication costs ofthis article were defrayed in part by page charge the actual compound that is detected in the urine of these patients payment. This article must therefore be hereby marked "advertisement" by this method is 7-biopterin. However, it is probable that the in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7-biopterin detected was derived from 7-BH4. 10109 Downloaded by guest on September 29, 2021 10110 Biochemistry: Davis et al. Proc. Natl. Acad. Sci. USA 89 (1992) as detailed (12). The hydroxylation of phenylalanine was Table 2. Comparison of the Km values for the aromatic amino monitored by either of two methods. The first measured the acid hydroxylases with BH4 and 7-BH4 as the cofactors formation of tyrosine (13); the second used a spectrophoto- Km (BH4), mM Km (7-BH4), mM metric assay (14). . Pure tyrosine hydroxylase was ob- Hydroxylase BH4 A.A. 7-BH4 A.A. tained from cultured pheochromocytoma PC12 cells (15, 16). Phenylalanine 0.003 0.25 0.16 0.06* The hydroxylation of tyrosine was measured by the 3H20 Tyrosine 0.3 0.01* 2.2 0.15 release method (16, 17). The phosphorylation of tyrosine Tryptophan 0.09 0.02* 1.7 ND hydroxylase by cAMP-dependent protein kinase was per- All apparent Km values were determined by computer-fit to the formed as described (18), under conditions that resulted in the Michaelis-Menten equation. Km A.A. is the Km value for phenylal- incorporation 0.6-0.7 mol of Pi per mol of enzyme subunit. anine, tyrosine, and tryptophan determined during their respective Tryptophan Hydroxylase. The hind and midbrain sections by their corresponding hydroxylases in the presence (including the superior and inferior colliculi and medulla of either BH4 or 7-BH4. ND, not determined because of insufficient oblongata) were dissected from whole rabbit brains and the levels of activity and high substrate inhibition. *Km value listed is really the S0.5 (the half-maximal substrate tissue was homogenized in 3 vol of 50 mM Tris-HCI (pH 7.5) concentration). Substrate inhibition prevented the determination of containing 0.1 M NaCl and 2 mM dithiothreitol. After cen- a reliable Km value. trifugation for 40 min at 110,000 x g (4°C), tryptophan hydroxylase was precipitated from the supernatant fraction 6MPH4 is the cofactor, catalysis proceeds at a rapid rate with by dropwise addition ofa solution of50% acetic acid to a final or without phenylalanine or lysolecithin preactivation. Sim- pH of 4.8. This mixture was stirred for 10 min at 4°C and the ilarly, these activation steps appear to have little effect on the acidified mixture was centrifuged for 10 min at 12,000 x g rate of catalysis when 7-BH4 is the cofactor; i.e., in this (4°C). The pellet was resolubilized in homogenization buffer; respect, the cofactor activity of 7-BH4 is similar to that of this preparation served as the source of tryptophan hydrox- 6MPH4 rather than BH4 (Table 1). Thus, the rates of the ylase used in these studies. The specific activity of this phenylalanine-dependent oxidation of BH4, 6MPH4, and enzyme preparation was -0.35 nmol per min per mg. Tryp- 7-BH4 by phenylalanine hydroxylase show only a small tophan hydroxylase activity was measured by the fluoromet- variance when the enzyme has been activated by either ric determination of 5-hydroxytryptophan (19). phenylalanine or lysolecithin, whereas the oxidation of BH4 is 20- to 35-fold slower when the enzyme has not been RESULTS activated (Table 1). The enzymatic oxidation of7-BH4 is =85% uncoupled (10), The Kinetic Properties of Phenylalanine Hydroxylase with indicating that the rate of tyrosine formation in the presence 7-BH4. Phenylalanine hydroxylase catalyzes the hydroxyla- of7-BH4 is =15% ofthe rate with BH4 for the fully activated tion of phenylalanine at a relatively slow rate in the presence enzyme.t On the other hand, under conditions where phen- of BH4 (14). This rate can be substantially increased by ylalanine hydroxylase has not been preactivated, the rate of preactivating the enzyme with its substrate, phenylalanine tyrosine formation with 7-BH4 as the cofactor is -3 times (20-22), or by exposure of the enzyme to certain phospho- faster than with BH4 as the cofactor. (23), such as lysolecithin (Table 1). In contrast, when The plot of the dependence of initial rate of tetrahydrop- terin oxidation on the concentration of 7-BH4 with phenyl- Table 1. Dependence of the initial rate of the phenylalanine- alanine hydroxylase follows classical Michaelis-Menten ki- dependent tetrahydropterin oxidation on the activation of netics with a Km value 60-fold higher than the Km determined phenylalanine hydroxylase for BH4 under comparable conditions (Table 2). In contrast, Activity, the analogous plot obtained by varying the concentration of ,umol per min Fold phenylalanine is complex (Fig. 1) and displays both cooper- Cofactor Activator per mg activation ativity at low phenylalanine concentrations (Inset) and sub- (6R)-BH4 None 0.52 1 strate inhibition at high phenylalanine concentrations. These Phenylalanine 12.4 24 kinetic characteristics were observed when the concentration Lysolecithin 18.3 35 of 7-BH4 was maintained at 0.6, 1.0, and 6.9 times its Km. 6MPH4 None 21.2 1 Because of the complexity of the plot, the kinetic constants Phenylalanine 22.8 1.1 can only be estimated. A minimum value for the maximum Lysolecithin 26.0 1.2 velocity of the enzymatic oxidation of7-BH4 is =5 t.mol per 7-BH4 None 10.5 1 min per mg and the concentration of phenylalanine required Phenylalanine 12.8 1.2 for half-maximal activity is 660 ,uM. This apparent Km for Lysolecithin 9.4 0.9 phenylalanine is -20%o the value found when BH4 is the Phenylalanine-dependent tetrahydropterin oxidation by phenylal- anine hydroxylase was measured spectrophotometrically. To test for tPhenylalanine hydroxylase requires a tetrahydropterin cofactor to the effect of phenylalanine activation, phenylalanine hydroxylase catalyze the hydroxylation of phenylalanine to tyrosine (24, 25). was preactivated at 26°C for 5 min with 1 mM phenylalanine prior to Since 1 mol oftetrahydropterin is oxidized per mol ofphenylalanine the assay. To minimize the effect of substrate inhibition on the hydroxylated when the cofactor is BH4, the rate of the hydroxy- reaction of phenylalanine hydroxylase with BH4 in the presence of lation reaction has usually been monitored for the oxidation of NADH during the NADH-dependent regeneration of the tetrahy- lysolecithin, the concentration ofphenylalanine in the assay was 0.23 dropterin by dihydropteridine reductase (14). A 1:1 stoichiometry is mM. All other assays with BH4 contained 1 mM phenylalanine. For also observed when the cofactor analogue 6MPH4 replaces BH4 in reactions containing 6MPH4, 5 mM phenylalanine was included, the reaction (14). However, substitution ofother cofactor analogues whereas when 7-BH4 was the cofactor 0.5 mM phenylalanine was for BH4 (e.g., 7-methyltetrahydropterin) can result in the uncou- included. The concentration of the cofactors BH4, 6MPH4, and pling of the hydroxylation of the substrate from the oxidation ofthe 7-BH4 were 100, 250, and 800 ,uM, respectively. With BH4, the cofactor (14). In these cases, phenylalanine hydroxylase partially concentration of the hydroxylase was 6.6 or 3.3 ,ug/ml for the behaves as a tetrahydropterin oxidase with the net oxidation of the unactivated and activated enzyme reactions, respectively. With tetrahydropterin exceeding the net hydroxylation of the substrate. 7-BH4 and 6MPH4, enzyme at 1.7 ,ug/ml was added. (6R)-BH4, The coupled and uncoupled oxidation of tetrahydropterins by 2-amino4-hydroxy-6-[dihydroxylpropyl-(L-erythro)-5,6,7,8-tetrahy- phenylalanine hydroxylase appear to proceed by alternative path- dropterin]. ways derived from a common enzyme-bound intermediate (26). Downloaded by guest on September 29, 2021 Biochemistry: Davis et aL Proc. Nadl. Acad. Sci. USA 89 (1992) 10111

6 ( 1004

80- 0 C

> 3 0 0.5 60 Phenylalanine, mM 40

C 20 bX 01. 0 0 6 12 0 25 50 Phenylalanine, mM 7-BH4 AiM FIG. 1. Dependence of the initial rate of the phenylalanine hydroxylase-catalyzed reaction on the concentration of phenylala- FIG. 2. Inhibition of nonactivated (curve a) and phenylalanine- nine with 7-BH4 as the cofactor. The assays were performed by activated (curve b) phenylalanine hydroxylase by various concen- monitoring the oxidation of NADH. V is the rate expressed in nmol trations of7-BH4. The assays were monitored for tyrosine formation. of NADH oxidized per min per mg ofenzyme. The concentration of Phenylalanine activation was performed as described in Table 1. The 7-BH4 was 760 AM and phenylalanine hydroxylase was present at 3.3 concentrations of phenylalanine and BH4 were 0.9 mM and 5 JM, ,ug/ml. (Inset) Saturation curve for phenylalanine at low substrate respectively. The 7-BH4 concentration was varied as indicated and concentrations. activated (4 jug/ml) or nonactivated (17 Ag/ml) phenylalanine hy- droxylase was added to the reactions. The data are expressed as a cofactor (Table 2). The plot of the dependence of initial rate percentage of the full activity in the absence of 7-BH4. The actual on phenylalanine concentration obtained from direct mea- amount of tyrosine formed during the 30-min assay was 70 and 250 surements of tyrosine production had essentially identical nmol for the reactions catalyzed by the nonactivated and the acti- characteristics to those in Fig. 1, except that the value of the vated enzyme, respectively. rate of hydroxylation is -15% that of the cofactor oxidation as described above. tyrosine (1.3 times the Km), is 7-fold higher than the corre- Inhibition of Phenylalanine Hydroxylase by 7-BH4. Despite sponding Km for BH4 with 20 AM tyrosine (2 times So.5, the the relatively high apparent Km for 7-BH4, this pterin mark- half-maximal substrate concentration). edly inhibits the phenylalanine-catalyzed conversion ofphen- The most dramatic effect of phosphorylating tyrosine hy- ylalanine to tyrosine when BH4 is the cofactor (Fig. 2). Even droxylase with cAMP-dependent protein kinase in vitro is a at a concentration as low as 1 ,M 7-BH4, an inhibition of >50%o is observed in the presence of5 AM BH4 and saturating phenylalanine. This inhibition increased as a function of 0 7-BH4 concentration. Preincubation of phenylalanine hy- 0 droxylase with phenylalanine to activate the enzyme only 0 slightly decreased the ability of 7-BH4 to inhibit the reaction (Fig. 2, curve b). On the other hand, increasing the concen- 0.

Y tration ofBH4 10-fold to 50 MM, (-20 times the K.) partially . alleviated the inhibition (Fig. 3, compare curve c with curve d), as did decreasing the concentration of phenylalanine OC- o =5-fold to 200 MM (Fig. 3, compare curve c with curve b). 0 Interestingly, a further decrease in the concentration of to phenylalanine to physiological concentrations (-60 1uM) ap- peared to entirely prevent the inhibition by 7-BH4 (Fig. 3, _ compare curve c with curve a). 0 The Kinetic Properties ofTyrosine Hydroxylase with 7-BH4. IL Substitution of 7-BH4 for BH4 in the tyrosine hydroxylase- dependent hydroxylation reaction of tyrosine changes some 0 25 50 ofthe kinetic properties ofthe enzyme (Table 2). Whereas the plot of the dependence of the initial rate on tyrosine hydrox- 7-BH4, UM ylation, with BH4 as the cofactor, is typically biphasic and FIG. 3. Effect ofincreasing the concentration of BH4 or decreas- displays severe substrate inhibition at tyrosine concentra- ing the concentration of phenylalanine on the inhibition of phenyl- tions >50 AM (16, 27, 28), with 7-BH4 as the cofactor, the plot alanine hydroxylase by 7-BH4. The assays were monitored for is hyperbolic with no substrate inhibition at concentrations of tyrosine formation. The concentration of7-BH4 was varied between tyrosine up to 300 MM (data not shown). Consequently, at 1 and 50 MM in samples containing 58 MM phenylalanine and 5 ,M high substrate levels, the rate of tyrosine hydroxylation with BH4 (curve a), 230 MM phenylalanine and 5 MM BH4 (curve b), 930 MM phenylalanine and 5 MAM BH4 (curve c), and 930 MM phenylal- 7-BH4 exceeds that measured with BR4 (data not shown). anine and 50 MM BH4 (curve d). Nonactivated enzyme at 40 ,g/ml The apparent Km for tyrosine in the presence of 7-BH4 is (curve a) or 10 ,g/ml (curves b-d) was added to each sample. The '13-fold higher than the half-maximal substrate concentra- data are the percentages ofthe full activities measured in the absence tion determined with the natural cofactor (Table 2). The of7-BH4, which were 3.8, 38, 83, and 68 nmol oftyrosine per 30 min apparent Km for 7-BH4, measured in the presence of0.2 mM for the conditions described in curves a-d, respectively. Downloaded by guest on September 29, 2021 10112 Biochemistry: Davis et al. Proc. Nati. Acad. Sci. USA 89 (1992)

120 B 7-BH4 as low as 1 substantially inhibited the reaction with BH4 even at concentrationsFM of BH4 equal to 2.5 times its Km. Increasing the BH4 concentration diminished the 100 inhibition of 7-BH4, implying that the inhibition is competi- tive. Decreasing the concentration of phenylalanine also { 80 80- alleviated the inhibition. In fact, at physiological concentra- 0 80 tions of phenylalanine (=60 ,M), even a 10-fold excess of 7-BH4, relative to BH4, failed to inhibit the hydroxylation XO 60 60- reaction (Fig. 3, curve a). Further studies are required to Is ~~~~~a determine whether the sensitivity of the 7-BH4 inhibition at ~40- 40 high phenylalanine concentrations is related to the substrate 0 inhibition observed in the absence of BH4. The cause of the hyperphenylalaninemia observed in pa- 20 b > 20 tients with an elevated excretion of 7-BH4 is still uncertain. Our results suggest that the potent inhibition ofphenylalanine 0- 0 hydroxylase by 7-BH4 may play a role in the abnormal 0.0 2.5 5.0 0.0 2.5 5.0 phenylalanine in these children. Although these 7-BH, mM patients have been classified as having mild hyperphenylal- aninemia, it should be noted that they have blood phenylal- FIG. 4. Inhibition of tyrosirie hydroxylase (A) and tryptophan anine levels between 5 and 20 times normal (31). Our in vitro hydroxylase (B) by increasing c:oncentrations of 7-BH4 in the pres- results suggest that low concentrations of7-BH4 would have ence of BH4. The data are pre.sented as the percentage of the fun little effect on the rate of phenylalanine hydroxylation in the activity (control) measured in the absence of 7-BH4. (A) Tyrosine liver when phenylalanine is present at normal physiological hydroxylase was either unactivaated (curve a) or activated (curve b) levels, whereas significant inhibition of enzyme activity by previous phosphorylation wiIth cAMP-dependent protein kinase. would occur at higher levels of phenylalanine. Therefore, In each case, BH4 was 100 AtM activities for the basal and phospIhorylatedenzymeswere25110and restricting the intake of phenylalanine of hyperphenylala- nmol per min per mg, respective ly. (B) Partially purifiled tryptophan ninemic patients who excrete high concentrations of 7-BH4 hydroxylase was assayed with thie indicated concentrations of7-BH4 in their urine might be expected to prevent 7-BH4 from in the presence of 100 AM BH 4 and 120 ,uM tryptophan. The fill inhibiting phenylalanine hydroxylation. Similarly, increasing activity in the absence of 7-BH4 was 0.33 nmol per min per mg. BH4 levels could also serve to counteract this inhibition, although to a lesser extent. This effect provides an explana- decrease in the apparent Km for BH4 (29). This phenomenon tion for the decrease in the blood phenylalanine levels of is believed to play a major roJleinthe regulationoftheactivity these patients when they are given BH4 (5). of the enzyme in vivo (29). VWhen the activity was measured The effect of 7-BH4 on the other two mammalian aromatic at pH 6, phosphorylation caused the expected decrease in the amino acid hydroxylases indicates that 7-BH4 is a weak Km for BH4 from 300 uM tUo 100 ,uM, without a significant competitive inhibitor and a poor substitute for the natural change in Vma. (data not shewn). A much more pronounced cofactor in both hydroxylation reactions. With tyrosine hy- effect, however, was observ,ed when 7-BH4 was substituted droxylase, the apparent Km for 7-BH4 is decreased by for BH4. The apparent Km foor 7-BH4 decreased from 2.2 mM phosphorylation and, therefore, increases competitiveness to =0.33 mM after phosphiorylation, without an apparent with BH4. Nonetheless, even after phosphorylation, the high change in V,,. (data not shoown). Accordingly, at physiolog- IC50 value for 7-BH4 suggests that under physiological con- ical concentrations of BH4 I[100 ,M (30)], the phosphory- ditions, low levels ofthis pterin should have little effect on the lation of tyrosine hydroxylaLse decreased the IC5o of 7-BH4 rate of tyrosine hydroxylation. Similarly, our preliminary from 2.5 mM to =0.48 mM (Fig. 4A). Increasing the con- results obtained with tryptophan hydroxylase suggest that centration ofBH4 to 250 AuM caused a corresponding increase the activity ofthis enzyme also would not be affected by low in the IC50 value to =700 julM, in agreement with the com- levels of 7-BH4 in vivo. Substantial inhibition of either of petitive nature of the inhibit:-ion by 7-BH4 (data not shown). these could result in neurological defects arising The Kinetic Properties oftTryptophan Hydroxyle with from the impairment of the synthesis of those neurotrans- 7-BH4. In the presence of 0.22 mM tryptophan, the rate of the mitters derived from tyrosine or tryptophan (4). Such defects tryptophan hydroxylase-catalyzed conversion of tryptophan have not, however, been reported in the hyperphenylalanine- to 5-hydroxytryptophan witii 7-BH4 is only 5% that with the mic patients with elevated levels of 7-BH4 (5, 31). natural cofactor (data not shown). The apparent Km for It is currently believed that the increase in excretion of 7-BH4 is 20-fold higher thaun for BH4 (Table 2). The corre- 7-BH4 is due to a genetic defect in 4a-hydroxytetrahydro- sponding Km for tryptophan iwith 7-BH4 as the cofactor could biopterin dehydratase (6, 7). The dehydratase is predomi- not be determined due to tihe low rate of catalysis for this nantly expressed in three tissues: liver, kidney, and the pineal reaction and significant suthstrate inhibition at tryptophan gland (32). Very little dehydratase activity, on the other hand, levels >0.2 mM. 7-BH4 iSs also a poor inhibitor of the was found in the brain, where tryptophan hydroxylase and hydroxylation of tryptophan by tryptophan hydroxylase with tyrosine hydroxylase are both expressed, or in the adrenals, BH4 as the cofactor. An IIC50 for 7-BH4 of 3.3 mM was where relatively high concentrations oftyrosine hydroxylase determined when the enzym e was assayed in the presence of are found.t Since 4a-carbinolamine, the proposed precursor

0.12 mM tryptophan and 0.11 mM BH4 (Fig. 4B).I of7-BH4, has been shown to be the product ofBH4 oxidation *The pineal gland contains a relatively high concentration of tryp- DISCUSSION tophan hydroxylase. Tryptophan hydroxylase from the pineal gland The potent inhibition by 7-BH4 of the phenylalanine hydrox- has several properties that differ from those of the brain enzyme. Thus, the finding of significant amounts of dehydratase activity in ylation reaction with BH4 as the cofactor was somewhat the pineal gland (32) suggests that further studies should be carried surprising in view of the finding that the Km for 7-BH4 is out to examine the kinetics of tryptophan hydroxylase from the 60-fold greater than that for the natural cofactor. However, pineal gland with 7-BH4; 7-BH4 may inhibit this enzyme more at saturating levels of phenylalanine, concentrations of strongly than it inhibits the corresponding brain enzyme. Downloaded by guest on September 29, 2021 Biochemistry: Davis et aL Proc. Natl. Acad. Sci. USA 89 (1992) 10113 by tyrosine hydroxylase during the enzymatic conversion of 7. Davis, M. D., Kaufman, S. & Milstien, S. (1991) Proc. Natl. tyrosine to dopa (33, 34), the low dehydratase activity in a Acad. Sci. USA 88, 385-389. tissue where tyrosine hydroxylase is found might result in the 8. Huang, C. Y. & Kaufman, S. (1973) J. Biol. Chem. 248, 4242-4251. formation of some 7-BH4. In normal individuals the amount 9. Kaufman, S. (1976) in Iron and Copper Proteins, eds. Ya- of7-BH4 that presumably would be formed in the tissues with sunobu, K. T., Mower, H. F. & Hayaishi, 0. (Plenum, New low dehydratase activity would likely be significantly less York), pp. 91-102. than the IC50 values determined for tyrosine and tryptophan 10. Davis, M. D. & Kaufman, S. (1991) FEBS Lett. 285, 17-20. hydroxylase and, thus, would not be expected to have a 11. Craine, J. E., Hall, E. S. & Kaufman, S. (1972) J. Biol. Chem. deleterious effect on either activity. 247, 6082-6091. The plot of the dependence of initial rate on phenylalanine 12. Kaufman, S. (1987) Methods Enzymol. 142, 3-17. 13. Udenfriend, S. & Cooper, J. R. (1952) J. Biol. Chem. 196, concentration with 7-BH4 as the cofactor deviated substan- 227-233. tially from a classical rectangular hyperbola, showing coop- 14. Kaufman, S. & Fisher, D. B. (1974) in Molecular Mechanisms erativity at low phenylalanine concentrations and marked ofOxygen Activation, ed. Hayaishi, 0. (Academic, New York), substrate inhibition at high phenylalanine concentrations pp. 285-369. (Fig. 1). The sigmoidal shape ofthe plot at low phenylalanine 15. Kuhn, D. M. & Billingsley, M. L. (1987) Neurochem. Int. 11, concentrations is similar to that reported for the analogous 463-475. plot with BH4 as the cofactor. For the natural cofactor, this 16. Ribeiro, P., Pigeon, D. & Kaufman, S. (1991) J. Biol. Chem. apparent cooperativity has been postulated to be due to the 266, 16207-16211. 17. Nagatsu, T., Levitt, M. & Udenfriend, S. (1964) Anal. Bio- need to activate phenylalanine hydroxylase with phenylala- chem. 9, 122-126. nine prior to catalysis and the ability of BH4 to inhibit this 18. Wang, Y., Citron, B., Ribeiro, P. & Kaufman, S. (1991) Proc. activation (21, 22). The sigmoidicity of the plot, therefore, Natl. Acad. Sci. USA 88, 8779-8783. suggests that 7-BH4 may also hinder the activation of the 19. Friedman, P. A., Kappelman, A. H. & Kaufman, S. (1972) J. enzyme by phenylalanine. Biol. Chem. 247, 4165-4173. Substrate inhibition with phenylalanine has been reported 20. Kaufman, S. (1970) J. Biol. Chem. 245, 4751-4759. (23), previously, but to our knowledge, this is the first time 21. Ayling, J. E. & Bailey, S. W. (1983) in Chemistry and Biology it has been observed in the absence of some form of phar- of , ed. Blair, J. A. (de Gruyter, New York), pp. of the enzyme. The substrate in- 363-367. macological preactivation 22. Shiman, R. & Gray, D. W. (1980) J. Biol. Chem. 255, 4793- hibition observed in Fig. 1 is also somewhat less marked than 4800. that observed previously in the presence of BH4 (23). For 23. Fisher, D. & Kaufman, S. (1973)J. Biol. Chem. 248, 4345-4353. example, the concentration of phenylalanine that allows the 24. Kaufman, S. (1958) J. Biol. Chem. 234, 2677-2682. highest rate of catalysis (0.5 mM phenylalanine) is approxi- 25. Kaufman, S. (1963) Proc. Natl. Acad. Sci. USA 50, 1083-1093. mately twice that reported under the other conditions (23). 26. Davis, M. D. & Kaufman, S. (1989) J. Biol. Chem. 264, Although with the natural cofactor each ofthese deviations 8585-85%. from classical Michaelis-Menten kinetics [i.e., sigmoidal 27. Shiman, R., Akino, M. & Kaufman, S. (1971) J. Biol. Chem. kinetics and substrate inhibition (14)] have been reported 246, 1330-1340. individually, to our knowledge, these two characteristics 28. Lloyd, T. & Kaufman, S. (1974) Biochem. Biophys. Res. Commun. 59, 1262-1269. have not been previously observed together. Thus, these 29. Kaufman, S. & Kaufman, E. E. (1985) in and Pterins, kinetic characteristics of 7-BH4 make it an ideal cofactor to eds. Blakley, R. L. & Benkovic, S. J. (Wiley, New York), pp. probe certain aspects of the mechanism of phenylalanine 251-352. hydroxylase. 30. Levine, R. A., Miller, L. P. & Lovenberg, W. (1981) Science 214, 919-921. 1. Jervis, G. A. (1947) J. Biol. Chem. 169, 651-656. 31. Dhondt, J. L., Forzy, G., Hayte, J. M., Guibaud, P., Rolland, 2. Jervis, G. A. (1953) Proc. Soc. Exp. Biol. Med. 82, 514-515. M. O., Dorche, C. & Andre, S. (1987) in Unconjugated Pterins 3. Kaufman, S. (1958) Science 128, 1506-1508. and Related Biogenic Amines, eds. Curtius, H.-C., Blau, N. & 4. Kaufman, S. (1983) Adv. Hum. Genet. 13, 217-297. Levine, R. A. (de Gruyter, New York), pp. 257-263. 5. Blau, N., Curtius, H.-C., Kuster, T., Matasovic, A., Schoedon, 32. 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