CHAPTER 2
Amino Acid Hydroxylase Inhibitors
Edith G. McGeer and Patrick L. McGeer
I. Introduction 45 II. Properties of the Enzymes 46
A. Tyrosine Hydroxylase 46 B. Tryptophan Hydroxylase 50 C. Phenylalanine Hydroxylase 54 III. In Vitro Inhibitors of the Hydroxylases 56
A. Catechols 57 B. Iron-Complexing Agents 63
C. Compounds Capable of Easy Oxidation/Reduction 63
D . Amino Acid Analogs 65 IV. In Vivo Inhibitors of the Hydroxylases 71
A. Catechols 71 B. Iron-Complexing Agents 73
C. Compounds Capable of Easy Oxidation/Reduction 74
D . Amino Acid Analogs 74 E. Miscellaneous in Vivo Inhibitors 83
V. Indirect Mechanisms of Inhibition 84 A. Direct Feedback (Product) Inhibition 84
B. Substrate Availability 85 C. Hormonal Influences 86 D . Indirect Feedback (Interneuronal) Inhibition 88
V I . Conclusion 89 References 90
I. INTRODUCTION
There are three principal reactions occurring in animals which involve the introduction of a hydroxyl group into the aromatic ring of an amino acid. These reactions are the hydroxylations of tryptophan to 5-hydroxy- tryptophan ( 5 - H T P ) , of phenylalanine to tyrosine, and of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). There are certain similarities in
4 5
46 E. G. MCGEER AND P. L. MCGEER
these reactions but important differences as well. An understanding of these similarities and differences is needed for an interpretation of the various actions of different types of inhibitors. In each hydroxylation molecular oxygen is required (1) as well as the same, or a highly similar, pteridine cofactor. The general reaction is presumed to be
hydroxylase
X H4 + A r H + 02 > A r O H + H20 + X H2
where X is a pteridine and ArH represents the amino acid substrate.
The reaction is in each case irreversible. Ferrous ion may be involved in all three hydroxylations, although the evidence in most cases rests mainly on the inhibitory action of some iron-chelating agents. Some nonenzymic oxidation of tryptophan (2) and phenylalanine (3) occurs in the presence of 02, Fe
2 +
, and a tetrahydropteridine.
Each hydroxylase is highly concentrated in a particular, and different, functional area of the body. Phenylalanine hydroxylase is highly specific to the parenchymal cells of the liver. Tyrosine hydroxylase is localized in chromaffin cells of the adrenal medulla, postganglionic nerve cells of the sympathetic nervous system, and dopaminergic and noradrenergic nerve cells of the brain. Tryptophan hydroxylase is found principally in the melatonin-producing parenchymal cells of the pineal gland, the serotonergic neurons of the raphe system of the brain, the argentaffin cells of the gut, and mouse mast cells.
Melanin formation also involves hydroxylation of tyrosine. The reac
tion takes place in melanocytes, which are originally derived from the neural crest. The enzyme involved, tyrosinase, is a copper-containing enzyme and is different from the tyrosine hydroxylase of the adrenal medulla or nerve cells. It may carry the reaction sequence further than DOPA ( 4 ) , although conversion of [
3
H]tyrosine to [ 3
H]DOPA in mouse melanoma in vivo has been taken as support for the postulate that DOPA is the physiological intermediate in melanin formation (5).
II. PROPERTIES OF THE ENZYMES
A. Tyrosine Hydroxylase
1. ASSAY
The most convenient methods of assay for tyrosine hydroxylase are radiometric, although one fluorescent method has been published (6).
One widely used technique is to measure the tritiated water released following conversion of 3,5-tritio-L-tyrosine to 5-tritio-DOPA ( 7 , 8).
2. AMINO ACID HYDROXYLASE INHIBITORS 47 One mole of singly tritiated water is formed for each mole of tyrosine that is converted to DOPA, and the water is separated by freeze-drying it into a counting vial or by passage through an ion-exchange column, which absorbs the tritiated amino acids.
Another method is to measure the [ 1 4
C ] D O P A formed during incuba
tion with [ 1 4
C]L-tyrosine. A DOPA decarboxylase inhibitor such as NSD-1034 (JV-methyl-Af-3-hydroxyphenylhydrazine) is employed to prevent further metabolism of DOPA. The [
1 4
C]DOPA is isolated on an alumina column, eluted, and counted (4, 9). Both methods are rapid and simple and employ tyrosine concentrations below saturation. Experi
ments with brain homogenates incubated with a mixture of [ 1 4
C ] tyrosine and tritiated tyrosine indicate that the two methods give entirely com
parable results (10). The tritiated tyrosine method gives higher blanks (and lower test to blank ratios) than the
1 4
C method, presumably be
cause of exchange of tritium between tyrosine and water molecules during the incubation and isolation procedures (10a).
2. LOCATION
The most active site of tyrosine hydroxylation in the body is in the adrenal medulla, which has an activity (Vmax) of the order of 1000 nmoles/hour X gm of wet tissue (9). This is about 10 times the activity of the caudate nucleus, the putamen, and the substantia nigra, which have activities at least five times those in any other brain area (11, 12). High activities are found in abnormal chromaffin tissue such as pheochromocytoma and neuroblastoma (13). Considerable activity is also found in tissue having a high concentration of sympathetic nerve endings (14) such as the superior mesenteric artery (15) and the heart.
The heart tyrosine hydroxylase activity is greatly reduced in congestive heart failure (16). The high-activity tissue from adrenals, brain, and heart has been used for most inhibitor studies.
The tyrosine hydroxylase in the chromaffin cells of the adrenal medulla can be easily solubilized (17-20a). The same is true of the tyrosine hydroxylase of the heart, vas deferens (21), or substantia nigra, but the enzyme in the caudate nucleus and putamen is much more firmly bound to particles (22, 23). Subcellular localization studies of caudate tyrosine hydroxylase have shown it to be highly localized to the nerve ending or synaptosomal fraction (24-27), as would be expected of an enzyme associated with synthesis of a neurotransmitter or neuromodula
tor. The substantia nigra contains cell bodies of dopaminergic neurons whose axons terminate in the caudate and putamen. Despite the different
48 E. G. MCGEER AND P. L. MCGEER
behavior of the enzyme in these different brain locations, it is neverthe
less associated with the same neuronal system. Presumably the enzyme is soluble in the soma of the cell where it is being synthesized by ribo- somes but becomes membrane bound when it reaches the synaptosomal areas (28).
3. PROPERTIES
The adrenal enzyme has been purified 400- to 1000-fold (17, 19, 28).
The enzyme purified 400- to 500-fold was reported to require a pteridine cofactor plus a thiol compound, molecular oxygen, and ferrous ions for maximal activity (Table I ) . Recently, using enzyme purified more than 1000-fold, Shiman et al. (19) have found that catalase can substitute for Fe
2+
ions and that either acts to protect the enzyme against inactiva- tion by the H202 generated by nonenzymic oxidation of tetrahydrobiop- terin. A synthetic pteridine, 5,6-dimethyltetrahydropteridine ( D M P H4) , is generally used for in vitro studies. High concentrations of D M P H4
( > 1 - 1 0 mM) or of Fe 2+
(>2.5 mM) are each inhibitory in some prepa
rations (29), but the inhibitory effect of excess D M P H4 may be lessened by adding Fe
2+
(8). Highly purified adrenal enzyme is not inhibited by excess cofactor (19). The effects of varying concentrations of 02 depend to some extent on the concentration of cofactor present (30).
Structural requirements for cofactor activity apparently include a 2-amino group, a 4-hydroxy group, and an unsubstituted nitrogen in the 5 position (8). As with liver phenylalanine hydroxylase, the order of activity of various pteridines tested is tetrahydrofolate < D M P H4 <
6-methyltetrahydropteridine < tetrahydrobiopterin (19, 31). The natural in vivo cofactor in the adrenal medulla has been reported to be tetrahy
drobiopterin (31, 32). This compound has been isolated from liver and is believed to be the endogenous cofactor for phenylalanine hydroxylase
(33), but its origin in the body is still obscure.
Exogenous cofactor does not stimulate the particle-bound tyrosine hy
droxylase in brain homogenates [cf. Table I (34-36), also 9, 22, 37], This may not, however, reflect a lack of need for cofactor. It could indicate that sufficient cofactor is already contained within the particles or that the exogenous cofactor is not taken up by the particles. Cofactor activity showing the chemical characteristics of tetrahydrobiopterin has been measured in brain and kidney as well as in liver (38), which sug
gests its ubiquitous nature.
The partially purified adrenal enzyme does not hydroxylate D-tyrosine, m-tyrosine, tyramine, or tryptophan. It does convert phenylalanine to
2. AMINO ACID HYDROXYLASE INHIBITORS 49
T A B L E I
RELATIVE ACTIVITIES OF A D R E N A L AND B R A I N T Y R O S I N E H Y D R O X Y L A S E PREPARATIONS IN A S S A Y SYSTEMS LACKING V A R I O U S COMPONENTS
Tyrosine hydroxylase source Partially purified
enzyme fro m adrenal Crude Crude rat adrenal brain Supernate Particulate homogenate homogenate
System (%Y ( % )
M
( % ) d
(7c) d
Complete system
e
100 100 100 100
Minus D M P H4 2 . 3 0 5 86
Minus D M P H4 and S H 5 109
Minus S H 38 14.5 107
Minus F e
2+
22 20 7 8 . 5 100
Minus F e
2 +
, SH, and
D M P H4 109
Minus 02 0 . 8 5 1 2
Minus enzyme 0 0 0
0
Data from 3J+.
b
Data from 35.
0
Recent studies (35a) suggest that the trypsin treatment used in the purification of particulate enzyme produces a tyrosine hydroxylase that is only a fragment of the native form. d
Data from 36.
e
Complete system contains D M P H4, 2-mercaptoethanol (SH), F e
2 +
, O2 of air, and enzyme.
tyrosine, suggesting that the enzyme has the capacity to hydroxylate in the para position if the meta position is not substituted (39, Jfl).
The highly purified adrenal enzyme is said to hydroxylate phenylalanine as rapidly as it does tyrosine if tetrahydrobiopterin, rather than D M P H4, is used as cofactor (19). The ability of tyrosine hydroxylase to accept phenylalanine as a substrate is one of the many significant interactions between the principal hydroxylating enzymes which may have some im
portance in vivo.
The liver is obviously the principal site for the normal conversion of phenylalanine to tyrosine for it is the liver phenylalanine hydroxylase that is missing in phenylketonuria (41). The small conversion of phenyl
alanine to tyrosine that has been observed in phenylketonuria has been attributed to tyrosine hydroxylase action rather than to any residual phenylalanine hydroxylase or to nonenzymic conversion (17).
5 0 E. G. MCGEER AND P. L. MCGEER
Tyrosine hydroxylase appears to be the rate-limiting step in catechol
amine synthesis. The Vnmx for tyrosine hydroxylase in catecholamine- synthesizing tissues is generally more than 100-fold lower than that for DOPA decarboxylase or dopamine /^-hydroxylase, the other two en
zymes involved in norepinephrine (NE) synthesis (34)- Some recent studies on DOPA decarboxylase activity in human brain tissue (42-43b) have indicated low levels of activity and suggested that this step may also be important in the regulation of catecholamine synthesis in human brain. It is probable, however, that the low values of DOPA decarboxy
lase so far measured in human brain tissues do not indicate the true in vivo activity. The lack of effect of inhibitors of DOPA decarboxylase or dopamine /^-hydroxylase {44) on central catecholamine levels is a strong argument for tyrosine hydroxylase being rate limiting for cate
cholamine synthesis and emphasizes the significance of metabolic inhibi
tors of this hydroxylase (45, 46).
The Km for tyrosine hydroxylase toward substrate has been variously reported as 4 - 1 0 X 10"
5
M for the adrenal medulla (28, 30, 34, 36), 2 X 10~
5
M for guinea pig heart (47), and 5 X 10"
6
M for crude rat brain homogenates (36). Values for tyrosine in the brain and adrenal medulla are of the order of 10~
4
M, a concentration that would fully saturate the enzyme. It is not surprising therefore that tyrosine loads do not have an appreciable effect on central catecholamine synthesis (34). Even in starvation, tissue tyrosine levels do not drop appreciably, which suggests that lowering tyrosine levels in the diet would also have little influence on the rate of catecholamine synthesis. The Km toward D M P H4 of the partially purified adrenal enzyme is about 5 X 10"
4 M U).
B. Tryptophan Hydroxylase
1. ASSAY
Attempts to assay tryptophan hydroxylase by release of tritium as in the tyrosine hydroxylase assay were unsuccessful because of a phe
nomenon known as the "NIH shift" (48). The tritium in 5-tritiotrypto- phan is not released to form water but instead shifts to the 4 position
on the indole ring (49). The 1 4
C method analogous to that used for tyrosine hydroxylase also presents problems. Measurement of the conver
sion of [ 1 4
C ] tryptophan to [ 1 4
C]5-hydroxytryptophan (5-HTP) is com
plicated by difficulties in separating the two amino acids.
2. AMINO ACID HYDROXYLASE INHIBITORS 51 The simplest method for determination of tryptophan hydroxylase is to measure the conversion of methylene-[
1 4
C] tryptophan to [ 1 4
C]sero- tonin in the presence of a monoamine oxidase (MAO) inhibitor to pre
vent conversion of the serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by the large amounts of MAO present in most 5-HT-forming tissues. The [
1 4
C]5-HT can be easily separated from the [ 1 4
C ] trypto
phan on an ion-exchange column (50, 51). This method presumes the presence of sufficient amounts of 5-HTP decarboxylase to convert completely all of the 5-HTP formed to 5-HT. While this is the case for most crude homogenates (51), it cannot be presumed to apply in cases where partially purified enzyme is used, where compounds are added that might inhibit 5-HTP decarboxylase, or where the tissues might lack adequate quantities of the decarboxylase. In such cases, the deficit of decarboxylase can be made up by adding a partially purified L-amino acid decarboxylase (52-54) or a rat kidney supernate which has virtually no tryptophan hydroxylase (52-54) but which has large amounts of an enzyme that can decarboxylate 5-HTP (51). Another method of tryptophan hydroxylase assay involves the formation of
[ 1 4
C ] 5-HTP in an assay system containing a decarboxylase inhibitor, its separation from [
1 4
C ] tryptophan by chromatography, and measure
ment of its radioactivity (54-56). A third radioactive method measures the amount of
1 4
C 02 formed from [l- 1 4
C]L-tryptophan and depends not only on the presence of excess decarboxylase but on the greater activity of the decarboxylase toward 5-HTP than toward tryptophan (57). Less sensitive methods involve fluorometric measurement of the 5-HT or 5-HTP formed (58-60).
2. LOCATION
The first reports of tryptophan hydroxylation illustrate the difficulties of cross interaction of hydroxylases and nonenzymic oxidation. A super
natant fraction from rat liver that required a pyridine nucleotide, oxy
gen, and a relatively high concentration of substrate (61), and a particu
late fraction from rat or guinea pig intestinal mucosa that required ascorbic acid and cupric ions but not oxygen (62), were reported to convert tryptophan to 5-HT. Further investigation showed that the ac
tivity in liver supernate was really phenylalanine hydroxylase, for which L-tryptophan is a relatively poor and physiologically unimportant sub
strate \63, 64). Doubt was cast on the intestinal mucosa system by the demonstration of nonenzymic oxidations in the presence of ascorbic acid and transition metals (65).
5 2 E. G. MCGEER AND P. L. MCGEER
A specific tryptophan hydroxylase has now been shown in a number of tissues in the following approximate order of activity: mouse mast cell tumor (52, 53, 58, 66), pineal gland (53, 56, 67), carcinoid tumor (53, 55), brain stem (63, 68, 69) and other brain areas (51, 70), and, to a much lesser extent, stomach and duodenum (53, 56). Human pineal has been shown to have activity (71). The combination of a low concen
tration of the hydroxylase in stomach and duodenum with a relatively high concentration of 5 - H T has been hypothesized to mean either an endogenous inhibitor or a low turnover rate. Brain and pineal hy
droxylase activities, on the other hand are closely parallel to the 5-HT distribution (51, 69, 71a).
The weight of evidence indicates that the majority of brain tryptophan hydroxylase is particle bound (51, 70) while that of the pineal gland, carcinoid tumor, or mouse mast cell tumor appears to be either primarily supernatant or else easily solubilized. There is one report that brain tryptophan hydroxylase, like tyrosine hydroxylase, is associated with nerve endings (71a). This is consistent with histochemical evidence for distinct serotonergic, dopaminergic, and noradrenergic neurons in brain with the amines being concentrated in the neuronal axons and nerve endings (72).
3. PROPERTIES
Although there has been considerable controversy with regard to the cofactor requirements of the enzyme, it appears that, in all its forms, it requires oxygen at a higher partial pressure than does tyrosine hy
droxylase. The supernatant tryptophan hydroxylase, whether prepared from pineal gland, carcinoid cells, brain stem homogenates, or mouse mast cells, requires exogenous D M P H4 together with 2-mercaptoethanol (Table II) (52, 53, 58, 71a, 73-75). The particle-bound enzyme, on the other hand, whether in brain (51, 54, 70, 71a) or in neoplastic mouse mast cells (58), is not activated by exogenous D M P H4. An early report that slices showed much more tryptophan hydroxylase activity than did homogenates was attributed to the presence of an endogenous cofac
tor that was released on homogenation and, at least in the case of pineal tissue, could be replaced by exogenous D M P H4 (56).
A requirement for Fe 2+
has been demonstrated only for mast cell en
zyme (75), but the inhibition of tryptophan hydroxylase from all sources by metal-chelating agents such as «,a'-dipyridyl suggests that Fe
2+
is generally involved. Ferrous ions are reported to stimulate the activity of tryptophan hydroxylase from guinea pig brain stem by 2 0 - 3 0 % when
2. AMINO ACID HYDROXYLASE INHIBITORS 53
T A B L E I I
TRYPTOPHAN H Y D R O X Y L A S E ACTIVITIES IN A S S A Y SYSTEMS LACKING V A R I O U S COMPONENTS
E n z y m e source
Mouse Brain stem
Beef Carcinoid mast soluble Crude rat System pineal ( % )
a
cells ( % )
a
cells ( % ) ° (%)
a
brain ( % )
b
Complete system
0
100 100 100 100 100
Minus D M P H4 9 0 6 4 98
Minus F e
2+
123 100 7 . 3 125 109
Minus 02 9 0 10
Minus S H 2 . 3 26 3 . 6 18 129
Minus D M P H4,
Fe
2
+ and S H 138
a
Data from 78.
b
Data from 51.
0
Complete system contains D M P H4, F e 2 +
, 02, 2-mereaptoethanol ( S H ) , and enzyme.
the Fe 2+
concentration is below 10 fiM but to be rather inhibitory at 100 pM (76).
The Km toward substrate of tryptophan hydroxylase has been vari
ously reported as about 1-4 X 10~
5
M for the enzyme from malignant mouse mast cells (52, 58, 66, 75) and from brain (50, 51). Lovenberg et al. (52) reported a Km toward tryptophan for the enzyme from beef pineal and rat brain of the order of 3-5 X 10~
4
M, but this seems high in view of the other data in the literature. The normal tissue content of tryptophan in brain is about 4 X 10~
5
M and is thus at best barely sufficient to saturate the enzyme. It is thus apparent why the 5-HT content of tissues varies with the tryptophan level in the diet (77-80), why it is increased in brain by intraperitoneal injections of tryptophan
(81), and why it is depressed by agents such as L-a-methyltryptophan that decrease brain tryptophan levels. The latter is presumably the result of stimulation of liver tryptophan pyrrolase (82). The substrate/enzyme relation for tryptophan hydroxylase is therefore quite different from that for tyrosine hydroxylase, where the tyrosine levels in tissue are normally saturating (see Section V,B).
Tryptophan hydroxylase does not hydroxylate D-tryptophan (54, 75>
83). Phenylalanine was initially reported to be a substrate for trypto-
54 E. G. MCGEER AND P. L. MCGEER
phan hydroxylase and this was suggested to have some implications in phenylketonuria (52). Later work with partially purified enzyme from brain stem (54, 58, 68) or carcinoid tumor (84), however, indicated that tryptophan hydroxylase does not hydroxylate either phenylalanine or tyrosine, although both act as competitive inhibitors. The data on enzyme purified from malignant mouse mast cells are conflicting, with one report (58) indicating that phenylalanine is not hydroxylated and another (75) suggesting that phenylalanine is an excellent substrate.
Tryptophan hydroxylase, like tyrosine hydroxylase, appears to be rate limiting in the synthesis of serotonin. Under normal conditions, there is more than a 100-fold excess of the L-amino acid decarboxylase (which decarboxylates both DOPA and 5-HTP) in 5-HT-forming tissues, and decarboxylase inhibitors do not have significant effects on 5-HT levels in vivo (46). This again points out the importance of metabolic inhibitors of the hydroxylases which are not only rate limiting but are the point of enzymic distinction between the 5-HT and catecholamine biosynthetic pathways.
C. Phenylalanine Hydroxylase
1. ASSAY
Phenylalanine hydroxylase is generally assayed by measuring the amount of tyrosine formed using the fluorometric method of Waalkes and Udenfriend (85) or the colorimetric method of Udenfriend and Cooper (86). Somewhat more sensitive radiometric procedures involve the hydroxylation of either f
1 4
C] phenylalanine or p-tritiophenylalanine.
In the
1 4
C method, the [ 1 4
C ] tyrosine formed is separated by paper chromatography and counted (87). During the hydroxylation of p-tritio- phenylalanine, the tritium is not lost to water but instead does an "NIH shift" into the meta position (48, 88), necessitating treatment of the 3-tritiotyrosine formed with an agent such as iV-iodosuccinimide to re
lease the tritium as water. The tritiated water is separated from the radioactive amino acids and counted, as in the assays for tyrosine hy
droxylase (89, 90). The assay is most conveniently done using synthetic D M P H4 as cofactor, in the presence of either 2-mercaptoethanol or dithiothreitol, and either ferrous ions or catalase to protect the D M P Ht from destruction and to minimize nonenzymic hydroxylations. Fre
quently, the assay systems also involve the use of a second, nonspecific liver enzyme (dihydropteridine reductase) plus TPNH to maintain the pteridine cofactor in the active, reduced form (91-93).
2. AMINO ACID HYDROXYLASE INHIBITORS 5 5
2. LOCATION
Phenylalanine hydroxylase seems to be specifically and uniquely lo
cated in the liver and is a supernatant enzyme. The small amount of hydroxylation of phenylalanine that can be demonstrated in brain is probably due to tyrosine hydroxylase (94).
3. PROPERTIES
Phenylalanine hydroxylase has been purified manyfold from a super- nant fraction of liver and has recently been obtained in forms that are 8 5 - 9 0 % pure (95). The partially purified enzyme requires a reduced pteridine cofactor and is stimulated by Fe
2 +
. Further evidence for a dependency on Fe
2+
is the inhibition by agents such as a,a'-dipyridyl and the reactivation with Fe
2+
of preparations inactivated by dialysis against a,a:'-dipyridyl (96). As with tyrosine hydroxylase, D M P H4 is commonly used as the exogenous cofactor but this is less active than 6-monomethyltetrahydropteridine or tetrahydrobiopterin (31, 32), which has been isolated from liver (33). The Km toward D M P H4 is of the order of 4 - 6 X 10~
5
M (87, 97).
The Km toward substrate has been reported to be of the order of 1-3 X 10~
4
M for rat liver enzyme (98) and 1-9 X 10"
4
M for human enzyme (87, 99), but determination was handicapped for some time by the oft-reported inhibition of the enzyme by excess substrate (86, 91, 98, 100). The maximum velocity in rat liver is of the order of 2 7 /mioles/hour X gm of tissue (98), which is some 5 0 - to 1000-fold higher than the maximum velocities found for tyrosine and tryptophan hy
droxylases in adrenal, brain, gut, and other tissues where they exist.
The specific phenylalanine hydroxylase appears to develop after birth (98,101, 102), although some have suggested that the lack of hydroxylase activity in newborn liver is due to a deficiency of cofactor rather than a deficiency of enzyme (97). Maximal activity in rats is reached at about 5 0 - 6 0 days of age (103). It is the specific phenylalanine hy
droxylase that is lacking in phenylketonuria (41), and some have sug
gested that the specific enzyme is also qualitatively or quantitatively abnormal in dilute lethal ( d y d
1
) mice (99, 104-106). Further investiga
tions, however, have indicated no abnormality in phenylalanine hy
droxylase in such mice if studied at an age ( 1 4 - 1 6 days) when the effects of starvation or a terminal state are avoided (107).
The partially purified enzyme does not hydroxylate D-phenylalanine, D-tryptophan (108), or tyrosine (17, Jfi, 91). It does, however, hy-
5 6 E. G. MCGEER AND P. L. MCGEER
droxylate (17, 40, 91) L-tryptophan, /?-2-thienylalanine, 2-, 3-, or 4-fluorophenylalanine (109), p-chloro- or p-bromo- (but not p-iodo-)
(109a) phenylalanine (giving the m-halotyrosine) (110), and p-methyl- phenylalanine (giving m-methyltyrosine and p-hydroxymethylphenyl- alanine) (111). The enzyme is clearly distinct from the nonspecific aryl hydroxylase or hydroxylases (112) in liver microsomes, which (a) detoxify foreign aromatic materials such as aniline or nitrobenzene (96, 118, 114) j (b) convert tyramine to dopamine (115), and (c) hydroxylate melatonin (116) and other indoles (117-119) in the 6 position. This 6-hydroxylation is believed to be the major route of melatonin metabo
lism (120-123) and is inhibited by chlorpromazine (124). The hydroxyl
ation of tyramine to dopamine is inhibited by desmethylimipramine or ^-diethylaminoethyl diphenylpropylacetate but not by amethopterin
(125).
Despite extensive work and the availability of purified enzyme, the full complexities of the phenylalanine hydroxylase system, and particu
larly the factors involved in keeping the cofactor in the active, reduced form, remain to be elucidated. The tyrosine and tryptophan hydroxylase systems may eventually prove to be equally complex.
III. IN VITRO INHIBITORS OF THE HYDROXYLASES The physiological importance of the hydroxylases and the availability of easy methods of assay have led to extensive screening of compounds as possible inhibitors of these enzymes. Less extensive work has been done with phenylalanine hydroxylase than with tyrosine or tryptophan hydroxylase but some inhibitors are known. The compounds that have high activity as inhibitors of any of the hydroxylases may be generally
classified into four groups.
A. Catechols
B. Iron-complexing agents
C. Quinones, ketones, and similar compounds capable of easy oxidation/reduction
D. Amino acid analogs
Other compounds have considerably less activity. The hydroxylases are all relatively insensitive to sulfhydryl reagents (such as mercuric chloride or p-chloromercuribenzoate), cyanide, and iodoacetate (9, 83, 126), al
though Gal et al. reported some inhibition of "mitochondrial" brain
2. AMINO ACID HYDROXYLASE INHIBITORS 57 tryptophan hydroxylase by either p-chloromercuribenzoate or cyanide
(54) and of liver phenylalanine hydroxylase by 10 -4
M p-chloromercuri- benzoate, BAL, or sodium borohydride (127). Petrack et al. (35) found purified adrenal tyrosine hydroxylase to be markedly inhibited on pre
incubation with mercuric chloride or p-hydroxymercuribenzoate. One re
port suggested that folic acid antagonists such as amethopterin or 2-amino-3-hydroxypteridine are good inhibitors of phenylalanine hy
droxylase (91), but others indicated little or no inhibitory effect on phenylalanine hydroxylase (10-24% inhibition at 10"
4
M) (59, 126), on tryptophan hydroxylase (54, 69, 83), or on tyrosine hydroxylase (9).
Since all three hydroxylations are oxidation/reduction reactions ap
parently involving molecular oxygen, ferrous ions, and the same or a highly similar pteridine cofactor, it may be expected that compounds in classes A, B, and C will be active against all three enzymes. More specific inhibition would be expected in class D .
A. Catechols
A number of o-catechols were shown by Udenfriend et al. (89) to inhibit tyrosine hydroxylase by competition with the pteridine cofactor.
The o-catechol function was necessary for activity; neither phenolic derivatives nor 3-methoxy-4-hydroxyphenyl derivatives showed any significant inhibitory action. Both the requirement for an o-catecholic function and the mechanism as competitive toward cofactor have been confirmed by others in various systems (128, 129). The inhibition of par
ticle-bound hydroxylases by catechols is not affected by adding pteridine cofactor (83), but this may be due to the failure of exogenous cofactor to be taken up by the particles. It has been suggested that some cate
chols, such as n-propyl gallate or a,/?,/?-trimethyl-DOPA (126), may have a dual effect and inhibit not only by competition with pteridines but by competition with ferrous ions as well. Johnson et al. (128) re
ported that inhibition of tyrosine hydroxylase by a-n-propyldopacet- amide (H 22/54) was markedly decreased in the absence of exogenous Fe
2 +
; the inhibition by various arterenones such as 3,4-dihydroxy-a:-di- methylaminoacetophenone was, however, not appreciably affected.
Since many catechols are easily oxidized to quinones, they may also act partly by nonspecific effects on the oxidation/reduction systems and thus be a special subgroup of the type of inhibitors discussed in Section III,C.
As might be expected, catechols active against tyrosine hydroxylase
T A B L E I I I
PERCENT INHIBITION BY REPRESENTATIVE CATECHOLS IN V A R I O U S A S S A Y SYSTEMS
a-n- M e t h y l - Concentration (m M) of Propyl- amino- a-
dopacet- aceto- Methyl- D o p a Sub In amide catechol D O P A L - D O P A mine Ref
Enzyme Source strate D M P H4 hibitor (%) (%) (%) (%) (%) erence
Tyrosine Adrenal 0.1 0 . 5 0.025 66 35
hydroxylase 0.05 1 0.02 50 39
0.05 1 2 52 50 39
0.1 'Pa 0.1 67 30 4
0.1 1.7 0.1 78 55 128
0 . 0 5 - 0 . 1 1 0 . 0 5 70 130
Brain stem 0.02 0 0.1 80 37
0.012 0.1 84 25 68 56 4
Whole brain 0.005 0 0.1 79 57 68 69 9 Tryptophan Mast cell 0.125 0 . 2 0.1 90 60 70 30 75 hydroxylase 0.06 0 . 5 0.005 23 65 128
0.1 0 . 2 0 . 5 90 50 66
Whole brain 0.01 0 0.1 51 27 60 47 83 Brain mito
chondria 0.01 0 0 . 7 58 54
Phenylalanine Liver 10
&
0 0.1 100 108
hydroxylase 10
6
0 0.001 50 50 108
10
b
3 0.003 68 47 53 126
10 3 0.003 58 33 126
10 3 0.1 47 126
1.5 0 0.01 50 59
1.5 0 0.1 10 10 59
0 . 5 0 0 . 5 < 5 0 50 > 5 0 131
° T, concentration 5 m M in tetrahydrofolate.
b
With tryptophan as substrate.
58
2. AMINO ACID HYDROXYLASE INHIBITORS 59 were also generally found to be active against tryptophan hydroxylase and, to the extent that they have been tested, against phenylalanine hydroxylase. The exact percentage of inhibition given by a particular catechol can be expected to vary with the assay conditions used. Some representative data on a few catechols that have been tested by a number of investigators in different systems are shown in Table III {ISO, 1S1).
Early reports on the inhibition of the hydroxylation of tryptophan by liver preparations indicated that almost all o-catechols were highly active (108, 132), but these studies, as pointed out in Section II,B, were using a nonphysiological substrate. More extensive explorations using tyrosine hydroxylase from brain or adrenals, tryptophan hydrox- xylase from brain or mast cell tumors, and phenylalanine hydroxylase from liver indicate that the nature of the substitution on the catechol is important to inhibitory activity. Acidic catechols, such as 3,4-dihydroxy- benzoic acid and 3,4-dihydroxyphenylacetic acid, tend to be relatively inactive against all three hydroxylases. The most potent derivatives seem to be those with a neutral side chain such as a-chloro-3,4-dihy- droxyacetophenone, ethyl /?-3,4-dihydroxyphenylpropionate, or a-n-pro- pyldopacetamide (H 22/54). A ketone group next to the ring appears to potentiate activity, perhaps because it adds to the length of the oxida
tion/reduction resonance system. Further ring substitution, as in 6-hy- droxydopamine or 2-methyl-a-methyl-DOPA, seems to reduce or elimi
nate activity. In optically active compounds, such as DOPA, the D isomer is much less active than the L isomer. Thus, all but three or four of the most active catechols so far found (Table IV) may be classed as side-chain-substituted derivatives of 3,4-dihydroxyphenylacetamide
(dopacetamide), 3,4-dihydroxyphenylethylamine, or 3,4-dihydroxy- phenyl ketone.
The compounds in Table IV are listed in the approximate order of their activity against brain tyrosine hydroxylase. It is evident that, in general, this order also holds for adrenal tyrosine hydroxylase and even, to a great extent, for tryptophan and phenylalanine hydroxylases.
The latter two enzymes, however, seem to be somewhat more vulnerable than tyrosine hydroxylase to certain of the less substituted derivatives such as catechol itself or a-methyldopacetamide.
Dopamine and norepinephrine are included in Table IV because they have been proposed as physiological feedback inhibitors (see Section V,A). Their in vitro effect, however, is not as dramatic as that of some of the other catechols.
The inhibitory action of apomorphine toward tyrosine hydroxylase is of some interest since this compound has been suggested as a thera-
T A B L E I V
INHIBITORY ACTIVITY OF VARIOUS CATECHOLS RELATIVE TO ACTIVITY OF METHYLAMINOACETOCATECHOL ( I ) OR a-PROPYLDOPACETAMIDE (II) °
Inhibitor
T y p e
6 Tyrosine hydroxylase Brain
K D A M P I P
Adrenal
I P I F I P I P I P
Tryptophan hydroxylase Brain
P
Mast cell
Phenyl
alanine hy
droxylase Liver P I P I P I P 11™
C5
o
Esculetin (6,7-Dihydroxy-
coumarin) X n-Propyl gallate X Dihydroxyphenyl ethyl ketone X 122
Chloromethyl dihydroxyphenyl
ketone X 116 Dihydroxyphenyl ketone X 115 Epinine (A
r
-methyldopamine) X 102 n-Propyldopacetamide
iV,iV-Dibenzylarternone Methylaminoacetocatechol 3,4-Dihydroxybenzaldehyde L - D O P A
a-Pyridylaminoacetocatechol 3,4-Dihydroxyphenyl a-methyl-
aminoethyl ketone
iV-Methylepinephrine X DL-Isopropylnorepinephrine X a-n-Butyldopacetamide X a-Isobutyldopacetamide X 3,4-Dihydroxybenzophenone X
a-Methoxydopacetamide X a-Methyldopacetamide X < 19 a-Hydroxydopacetamide X
X X X X X
100 100 100 100
96
88 81
76 76
71
78 77
15 140
76 70
83 71 56
100 100
45
63
190 176 173 107
100 146 115
125 100
92 77
78
100 101
36 100 100 100 100
57
90 80 80
Aminoacetocatechol X 63 42 111
1-Piperidinoacetocatechol X 59 115 1-Pyrrolidinoacetocatechol X 59 128 Dopamine X 87 67 57 92 33 10
Dimethylaminoacetocatechol X 51 116
Cobefrin (a-methyldopamine) X 73 106 95 Ethyl 3,4-dihydroxyphenyl-
propionate X 73 130 Apomorphine X 73
3,4-Dihydroxyacetophenone X 50 5-Hydroxyarterenone X 50
a-Ethyldopacetamide X 50 90 tt-Isopropyldopacetamide X 48
DL-N-Ethylnorepinephrine X 72 107 L- « - M e t h y l - D O P A X 72 20 30 54 56 67 10 17 L-Epinephrine X 59 56 75
Isopropylaminoacetocatechol X 57 49 93 100
L-Norepinephrine X 49 30 50 55 72 10 55 a-Methylnorepinephrine X 47 49 Catechol X 45 49 6 158
a
The activities of each compound are expressed as a percentage of the activity in the same assay system of either methylamino
acetocatechol (I) or a-n-Propyldopacetamide ( H 2 2 / 5 4 ) ( I I ) ; a figure of 50 under a column labeled I would mean that particular compound was half as active an inhibitor as methylaminoacetocatechol. b
Active catechols are classifiable into derivatives of
HO HO
X (K)
and other ( M ) . c
Data from 9, 83.
d
Data from 4-
e
Data from 128.
f Data from 39.
° Data from 130.
h
Data from 35.
i
Data from 83.
* Data from 66.
(D) HO
?1 ?2
CH—CH—N (A) Y
k HO
Data from 75. 1
Data from 59.
m
Data from 126.
61
T A B L E V
PERCENT INHIBITION B Y VARIOUS COMPLEXING A G E N T S IN in Vitro A S S A Y S OF AROMATIC A M I N O A C I D H Y D R O X Y L A S E S
0
Inhibitor
Phenylalanine hydroxylase
Liver 0.1
&
l
c
0.1
c
Tyrosine hydroxylase Brain Adrenal
Tryptophan hydroxylase Brain
mito- Brain chondria 0 . 1
d
0 . 1 ' 0.1" 0.1
A
0.1' 0.01* 0.4'.* 0.1* 0.6™ 1.2*
4-Isopropyltropolone 8-Hydroxyquinoline 5-Iodo-8-hydroxyquinoline Bilirubin
o-Phenanthroline
2,9-Dimethy 1-1,10-phenanthroline a-a'-Dipyridyl
Desferrioxiamine
2-(4-Thiazolyl)benzimidazole Dibenzo [/,/i]quinoxaline 2,4,6-Tripyridyl-s-triazine Ethyl 3-amino-4fl
r
-pyrrolo- isoxazole-5 (6i/)-carboxylate a-Nitroso-/3—naphthol
Disulfiram
Sodium diethyldithiocarbamate Ethylenediaminetetraacetic acid
13"
24" 79 82
79"
49"
42"
33 26"
86 47 51
31
28
< 1 5
< 1 5 100
90 90
60 52
49
50
< 1 5 50 35
57 39
28
60 73
< 1 5
80 33
70 31
< 1 5
56 85
° Value above each column indicates millimolar concentration of inhibi
tor; values within table indicate percent inhibition b y inhibitor. b
Data from 108
c
Data from 126.
d
Data from 9.
e
Data from 8.
f
Data from 135.
0
Data from 136.
h
Data from 4.
* Data from 137.
> Data from 76.
k
Supernatant fraction. 1
Data from 83.
m
Data from 54.
n
Tryptophan as substrate.
62
2. AMINO ACID HYDROXYLASE INHIBITORS 63 peutic agent in Parkinson's disease (133). Its usefulness would presum
ably depend on stimulation of dopaminergic receptors and might be lessened by any inhibition of tyrosine hydroxylase. The inhibitory action of apomorphine is decreased by increasing concentrations of D M P H4 in the incubation medium (134) •
B. Iron-Complexing Agents
As discussed in Section II, stimulation of the hydroxylases by ferrous ions has been observed in only a few preparations, and the supposition that these enzymes are Fe
2+
dependent rests mainly on the inhibition generally observed with iron-complexing agents such as a,a'-dipyridyl and o-phenanthroline. A list of compounds that have been reported to inhibit substantially one or more of the hydroxylases and whose struc
tures suggest that they may act by complexing with ferrous ions is given in Table V (135-137). The potency of some of these agents seems well established but that of others such as ethylenediaminetetraacetic acid and sodium diethyldithiocarbamate is controversial, and the weight of evidence suggests little or no activity.
The chelating agents as a group do not appear as active as the cate
chols discussed in Section III,A. Partial reversal of the inhibition in vitro on addition of Fe
2+
ions to the incubation medium has been shown for o-phenanthroline (137), for ethyl 3-amino-4#-pyrrolo[3,4-c]isox-
azole-5(6AT)-carboxylate, which is the most active of a series of pyr- roloisoxazoles tested as inhibitors of adrenal tyrosine hydroxylase (136), for a,a'-dipyridyl (76), and for 4-isopropyltropolone (135).
C. Compounds Capable of Easy Oxidation/Reduction
A number of quinones, ketones, and related compounds (Table VI) (138-140) have been found to inhibit tryptophan, tyrosine, and/or phenylalanine hydroxylase but do not appear to be truly competitive with substrate, cofactor, or Fe
2+
ions (29, 138, 141)- It has been specu
lated that these compounds act by nonspecific interference with the oxi
dation-reduction processes. Such an interference might be expected in view of the quinoidal nature of the cofactor product. The strong inhibi
tion of tyrosine hydroxylase [Ki = 3.6 X 10"
7
M (29) ] by the quinoidal antibiotic, aquayamycin, could be partially reversed by addition of Fe
2+
but was increased [as was that of some simpler quinones (140)] with
T A B L E V I
PERCENT INHIBITION BY VARIOUS COMPOUNDS CAPABLE OF E A S Y O X I D A T I O N / R E D U C T I O N IN in Vitro ASSAYS OF AROMATIC A M I N O A C I D H Y D R O X Y L A S E S
0
Phenylalanine Tryptophan hydroxylase Tyrosine hydroxylase hydroxylase
Brain
Liver Brain Adrenal stem Brain Inhibitor 0.3* 0.08
c
l
d
>
e
0.V 1* 0.1* 0.00037* 0.001''* 0.1*-<
Aquayamycin 50 78 l,2-Naphthoquinone
m
98 50 1,^Naphthoquinone™ 70 l,3-Dichloro-l,4-naphthoquinone
m
55
2-Methyl-l,4-naphthoquinone 61 Menadione (vitamin K ) 50
2,5-Dimethoxybenzoquinone 75 6-Amino-7-chloroquinoline-5,8-quinone 79
1,8-Dihy droxy-4,5-dinitroanthraquinone 47
Adrenochrome 42 33 Sodium indophenol 54 55 2,6-Dichlorobenzoindophenol 80 73 3-Hydroxy-2-naphthaldehyde 87 < 2 0 Phenylpyruvic acid 55 42
p-Hydroxyphenylpyruvic acid < 5 47 20 < 1 5
2,5-Dihydroxyphenylpyruvic acid 68 < 1 0 p-Chlorophenylpyruvic acid 55
Indole-3-pyruvic acid 54 < 1 5 Af,V-Dimethyl-p-phenylenediamine 76 63 A^,iV-Diethyl-p-phenylenediamine < 1 5 75 Methylene blue 43 73 Azur eosin (Giemsa stain) 36 68
*S-Methylcysteine 56
a
Value above each column indicates millimolar concentration of inhibitor; values within table indicate percent inhibition b y inhibitor. b
Data from 99, 138.
c
Data from 138.
d
Data from 132.
e
With tryptophan as substrate.
* Data from 73.
o Data from 139.
h
Data from 140.
*' Data from 29.
> Data from 76.
k
Supernatant.
1
Data from 83.
m
Corresponding diols inactive (138).
64
2. AMINO ACID HYDROXYLASE INHIBITORS 65 increasing cofactor concentrations. These findings suggest that such quinones might inhibit in the same way as do high concentrations of cofactor (29). The inhibition of tryptophan hydroxylase by aquayamycin was almost eliminated by addition of dithiothreitol to the medium, sug
gesting interference with an oxidation-reduction system (76).
Some of these compounds, such as sodium indophenol and 2,6-dichloro- benzoindophenol, have shown approximately equal activity toward tyro
sine and tryptophan hydroxylases, but others have shown marked differ
ences in activity toward the two enzymes in the in vitro systems used.
This may be largely a matter of pH effects on unstable compounds.
Thus, for example, pyruvate derivatives such as indole-3-pyruvic acid and 2,5-dihydroxyphenylpyruvic acid were very active against tyrosine hydroxylase in vitro at pH 6.2 but had only minimal action against tryptophan hydroxylase at pH 7.8 (83). Inhibition of phenylalanine hydroxylase by phenylpyruvic acid and other "abnormal" oxidative me
tabolites of phenylalanine is not great enough to be of significance in phenylketonuria (141)-
Some substituted p-phenylenediamine derivatives are included in this category of inhibitors as well as methylene blue and azur eosin, each of which has a dialkylamino group in a position para to the nitrogen on the phenothiazine ring system. They may thus be considered analo
gous to substituted p-phenylenediamines. These compounds should all be capable of easy oxidation to a free-radical semiquinone form. Inhibi
tion of yeast growth and of certain enzymes such as succinic dehydro
genase by iV,Af-dimethyl-p-phenylenediamine has previously been at
tributed to such easy oxidation (11+2). As illustrated by this example, it may be expected that inhibitors of this class would affect a wide variety of oxidative enzyme systems.
D. Amino Acid Analogs
The most important group of inhibitors of the hydroxylases so far discovered is comprised of certain substituted derivatives of phenyl
alanine, tyrosine, and tryptophan. Table VII (143-147) lists most of the compounds of this class that have been found to show considerable inhibitory activity toward any one of the hydroxylases, while Table VIII (148-151) indicates the percentage of inhibition found for a few
aromatic amino acids in various in vitro assay systems. It is evident that the reported results are quite discordant in some cases as are, for example, the data for 3,5-diiodotyrosine. More detailed studies with 3,5-
T A B L E V I I
PERCENT INHIBITION OF AROMATIC AMINO ACID HYDROXYLASES FOUND in Vitro WITH SOME AMINO ACID A N A L O G S
0
Tyrosine hydroxylase Tryptophan Phenylalanine Adrenal Brain hydroxylase, brain hydroxylase, liver
Inhibitor 0.1
b
0 . 1
c
0 . 2
d
oT
e
OJL' 1* I* V Phenylalanine derivatives
a-Methyl-DL- 3-Iodo-a-methyl-DL- 3-Iodo-DL-
3-Bromo-DL-
4-Fluoro-a-methyl-DL- 4-Amino-a-methyl-DL- 3-Bromo-o;-methyl-DL- 4-Fluoro-DL-
4-Chloro-a-methyl-DL-
L_y
3- Hy droxy-a-me t hy 1-L- 2-Fluoro-DL-
/S-Methyl-DL- 4-Chloro-DL- 2-hydroxy-DL- 3-Hydroxy-DL- 3-Chloro-a-methyl-DL- 4-Iodo-DL-
3-Iodo-o-methoxy-DL- 4-Nitro-DL-
Tyrosine derivatives 3-Iodo-a-methyl-DL- 3-Iodo-L-
a-Methyl-L-
86
85 77 75 71 30 62 60 41
48
94 90
46 84 68 41
76 32 54 0 30
52
84 68 56
82 70 33
32
64 77 88 58
78 64 41 < 1 5
78 0 51 31
50 71 31
50 63 49 69
16
47 78 20
43 90
< 1 5 80
97 36 100 < 1 5
< 5 0
< 5 0
< 5 0
< 5 0
66
3,5-Diiodo-L- < 1 5 81 33 2-Fluoro-<*-methyl-DL 95
2-Chloro-a-methyl-DL- 84 3-Bromo-a-methyl-DL- 73
2a-Dimethyl-DL- 69 < 1 5 < 1 5
3-Amino-L- 65 < 1 5
3-Nitro-L- 64
a-Ethyl-DL- 60
3-Chloro-a-methyl-DL- 60 3-Fluoro-L- 50 ryptophan derivatives
a-methyl-5-hydroxy-DL- 80
5-Iodo-DL- 100 42
5-Bromo-DL- 98 45
5-Chloro-2-methyl-DL- 96 23
5-Chloro-DL- 90 44
5-Methyl-DL- 75 30
2-Methyl-DL- 68 28
5-Hydroxy-DL- 30 59 < 2 0
L-> 20 58
a-Methyl-DL- 17 52 27
6-Methyl-DL- 45 53
5-Fluoro-DL- 42 50
6-Chloro-DL- < 1 5 75
6-Fluoro-DL- < 1 5 80
° Value above each column indicates millimolar concentration of inhibitor; values within table indicate percent inhibition b y inhibitor. b
Data from 1^0.
c
Data from 143.
d
Data from 144, 145.
e
Data from 9, 146.
f D a t a from 83, 147.
0 D a t a from 126.
h
D a t a from 145.
l
' D a t a from 131.
1 Unsubstituted C o m p o u n d .
67
T A B L E V I I I
PERCENT INHIBITION OF AROMATIC A M I N O A C I D H Y D R O X Y L A S E S B Y SOME A M I N O A C I D A N A L O G S IN V A R I O U S in Vitro A S S A Y SYSTEMS
Concentration (mM) of Phenylalanines (%) Tyrosines (%) Tryptophans (%)
Enzyme Source Sub
strate DMPH4 Inhi
bitor L - D- 4-F- 4-C1- L - 3-1-
0.1 1 0.01 85
0.1 0.5 0.025 83
0.2 1 0.1
0.1 0 0.1 30 75 41 90
0.1 2 0.05
0.1 0 0.05
0.1 b 0.1 10 0 61
0.01 5 0.1 100*
0.05 b 0.02
0.05 2 0.2 54< 33 30
0.005 0 0.1 78 64 50 97
0.012 b 0.1 78 7
0.008 0 0.1 80 80
0.02 0 0.1 60
0.01 4 0.1
0.01 0 0.1 64 77 69 27 36
0.009 0 0.08 56
0.009 0 0.04 Activate
0.125 0.2 1 30
0.125 0.2 0.1 55
0.3 0.07 0.5 50«
10 3 0.1 0
0.43 0.09 5
16* 0 0.1 22 47 2
0.12 1 0.01 0
10* 3 0.1 14 67 14
10* 3 1
10 3 1 88
20* 0 0.1 80 39
20* 1 0.1 8 1
1 1 1 58 31
16* 0 0.2 40 0 32
16* 0 0.1 39
3.2 0 0.1 0
l - d - 5-F- 5-HO- =-Me- Ref-
erence
Tyrosine
hydroxylase Adrenal
00
Tryptophan hydroxylase
Phenylalanine hydroxylase
Brain
Heart Brain Mitochondria Mast cell
Liver
72 67
67 94 60
< 1 0
35
0 19
< 1 5
5-15 50
33 20
50 65
52
< 2 0
30«
40 85 US HO HO HO 4 148 89 H4,14$
9.H 4 23 87 HS 83, H7 54 54 75 75 149 126 149 108 40 126 126 126 132 182 145 150 151
0 151
Noncompetitive with substrate.
b
Concentration 5mAf in tetrahydrofolate.
c
Competitive with substrate.
* With tryptophan as substrate.
