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The roles of folate and pteridine derivatives in neurotransmitter metabolism
THE ROLES
OF FOLATE AND PTERIDINE DERIVATIVES NEUROTRANSMITTER METABOLISM
IN
ANTHONYJ. TURNER Department of Biochemistry, University of Leeds, 9 Hyde Terrace, Leeds LSZ 9LS. England The biologically active folate coenzymes are the derivatives of t~trahydrofolic acid (FH4) that function in the transfer of l-carbon groups in a variety of reactions in purine, pyrimidine and amino acid metabolism [I]. The ptcridine moiety, in addition to forming an integral part of the folic acid molecule. is also required as a donor of reducing equivalents in several amino acid hydroxylation reactions. A number of defects in both folate and pteridine metabolism are known to affect neurological function, suggesting an essential role for these compounds in the maturation of the nervous system and in synaptic dynamics [Z-4]. This review will concentrate on aspects of the metabolism of these compounds of current relevance to neuro~he~nistry and, in particLllar, to the metabolism of amino acid and amine ne~lrotransmitters. Such studies have been timited in the past because of the low concentrations of folatc derivatives in tissues. their instability and problems associated with their analysis. ~~~t~loil~~l r~~dioirnrn~~o~say procedures are now availabic for foiate measurements. The structures of folates and pteridines and the biochemistry of onecarbon transfer reactions have been described in detail elsewhere [I], although pathways relevant to this essay are shown in Fig. 1. Throughout this article the term ‘pterin’ will be used to refer to derivatives of the naturally occurring 2-am~~to-4-fiydroxypter~dine. as suggested elsewhere [I]. The role of folates in nucleic acid l~etabolis~l in brain will not be discussed here. Folatr ~?~~f~~~~~isi?l irr bruin. Although the synthesis of the pteridine ring system has been extensively studied in bacterial and insect systems. there is little information available concerning the biosynthesis of pterins in mammalian systems. However, it has been established that various mammalian tissues, including brain, possess the capacity to synthesize biopterin from GTP [S], and neurobiastoma cell lines have been shown to effect a similar synthesis [6]. The pathway of rnarnrn~~ljan pterin synthesis is presumed to be similar to that found in microbial systems. In contrast nialn~lal~a~ tissues do not have the capacity to synthesize folate derivatives and for human beings there is a daily dietary requirement of about 5Opg folic acid [Z]. The folic acid molecule is composed of a pterin linked through a ~?-amilloben~oyl moiety to one or more glutamate residues in T-carboxyl linkage (folate mono- and oligo-glutamates). 5-Methyltetrahydrofolate (CH,FH,) is the major form of folate present in serum and peripheral tissues BSwell as in the developing brain. However this is apparently not the case in the mature brain where almost SO per cent of the
total folate is present as FHJ oligoglutamat~ [4,7]. Folate levels in brain and cerebrospinal fluid are 2--3 times higher than those found in serum even in states of folate deficiency [S]. This indicates the importance of these compounds to the brain and suggests the existence of a regulatory mechanism for maintaining brain foiate levels. CH3FHa exists predominantly in a protein bound form in blood [9] and is concentrated into brain by a saturable uptake mechanism specific for folates [IO]. This requirement for the transport of reduced folates from blood is necessitated by the absence in brain of significant quantities of dihydrofolate rcductase [l If. Form~minotransfer~e, an enzyme which can contribute to the one-carbon pool in liver is also apparently absent from brain [12], suggesting that serine is the major endogenous source of one-carbon units in this tissue through the action of serine l~ydroxymetbyitransferase. Ail the enzymes required for the & IIOLW synthesis of ~-adenosy~methionine from serine are known to occur in brain (see Fig. 1). A number of studies [I2 -161 have examined the subcellular distributions of folate-metabolizing enzymes in nerve tissue and although there is general agreement on the cytosolic location of methylene FE& reductase, rn~t~ionin~ synthetase and methionine adenosyi transferase, discrepancies are apparent in the reported distribution of serine bydroxymethyitraI~sfer= ase. Davies and Johnston [13] and McClain et si~nificallt proportions &. [12] have reported (20-30*,,) of serine hydroxymethyltransferase in the FH4
ItGlycine
Methyl&FH,
4
Serine
HCHU
X
11’ CH, FH4 2
Homocysteine
j
A-W
w 4-
PPP;
-IS-Adenosyl
methibnine
Fig. 1. ~~t~rc~r~~~rsi~n of FH, derivatives in relation to amino acid and amine metabolism. The principal enzymes involved are: (1) merhylene-FH, reductase (EC 1.1.1.68); (2) CHIFHl homocysteine methyltransferase (EC 2.1.1.13); (3) methionine sdeaosyltransferase (EC X.1.6); (4) serine hydroxymethyltransferase (EC 2.1.2.1). The association of FH, with formaldehyde is presumed to be non-enzymk
cytosol. wilh the majority of lhe remaining activity occurring in the mitochoncirial fraction as in liver. In contrast. other workers hate conoi~idcct tktt this enzyme occurs almost cxclitsively in tnit~~~ll(~l~~~ri~ with negligible activity being present in the cytosol [14 Ih]. The dis~rcp~tt~~ies may reflect the d&rences in ~~o~~o~~~~is~tti~~~~ and fr~t~ti~~n~ttiot~conditions emplo>cd in these studies. A careful re-il~~~sti~~ti[~n of the l~~~~tlis~~ti~)tlof serinc hytfroxymetl~yltransfer3se in brain is inlp[~rt~ttl~ because of its possible involvement in a one-cnrbon shuttle system across the inner tnitochondrial mcmhrane [ 173. which would require both cytosolio and mitochondrinl forms of the emyme. Although a considerable proportion of cellular folates are present 21s their olipoglutamate derivatives. most studies on folate-metaboliziiig enzymes have tended to use the monoglutamates as substrates because of their ready ~tv~ii~tbility. However the oli~~~~lut~rn~~e forms of the f&tcs have been shown to be better substrates than the n7onoglLttalTintes for 21 number of folatc_iiitercctnvLrtit~~ enzymes [ 18 Xl]. Thus the oiigoplutamnte derivatives may not only represent storage forms or the folatcs as prcviottsly suggested [I]. but tnay function both as natural substratcS and as regulators of intr~~~ellul~tr folate-dependent reactions. Such possibilities should bt: borne in mind in studies on the enzymology of folate motabolistn. 7-/W lolc f!!’ ptefYrVi irr the S\‘lifhL’Si.\ ‘ji hic&gi’ltic* ~tt~ti~s. nteprtmary and rate-limiting step tn the svnthesis of the catecholamines involves the convct&n I..-tyrosi no r-?.4-dihydrosSiphcnyl3latiille of to (DOPA) by the myne tyrosine hydrosqlasc [Zl]. This enzyme catalyses a 3-substrate reaction involving tyrosine, oxygen and 3 reduced plerin cokictor. (probably telrah?idrohiopterin, B&i as shown in reaction (1): I.-tyrosinc
+ BH,
-C 0, ----)DOPA
BH,
+ NADH
+ BH,
“r H” ---;‘BH,
+- HI0
+ NAD
(1)
(2)
Regeneration of the reduced ptcrin is carried out by a pyridinc nucleotidc-depelidcnt enzyme. dihydropteridine reductase [12] (reaction 2). Phenyialaninc [3] and tryptophan hydroxytascs [1?4] also use ptcrin cofactors for the donation of reducing equivalents and similarly require pteridinc reductasc for regenerating the reduced cofactor. The related enzyme dtliydr~~folate rcductasc prob~tbl~ plays no role in the supply of Rtt, [25]. The re~Lt~~ttioi~ of ~tte~l~~~l~~rninesynthesis occurs mainly at the tyrosine hydroxylase step and may involve long-term modifi~~~io~~s of the amount of tyrosinc hydroxylase protein (tr~tns-syn~ptic indu~ti(~n) ;Ind short-term regulatory proccsses independent of changes in enzyme levels [Xl. This review will consider only the latter mechanisms, It is now well cstahlished that dopamine and noradrenaline inhibit tyrosine hydroxylase competitively with respect to the ptcrin cofactor [I??). Such ohservations suggest that release of catecholatnines from the nerve ending may result in an increase in the rate of amine synthesis due to the relief of end-product inliibi~ion of tyrosinc hydroxylase. Indeed, several studies have dernl~lis~~~ted an increase in the rate of
The synthesis of a number ol’ ncurotratismittcr~ including the catccholamincs may he regulated by prccursor availability (for disc&on see ref. 14). For example, elaation of tyrosine levels can bring about a transient incrensc in catccholaniinc synthesis [3S] and it has also been proposed that the levels of reduced ptcrin cofactor may regulate tJrosinc hydro~yl~ttion and amine synthesis [IS]. This suggestion is s~st~tin~d by the ~~erno~i~~r~iti~~rithat ir~tr~~~cli~ri~ula irtjection of bi~~pteriI1 [3’7j or its addition to sympathetic nerve tissue in culture [3X] or ~~n~tpt(~~~~t~~~l preparations [39] rcsitlls in ai increoscd conversion tyrosine to catecholnmincs. of an inhibitor of pteridinc rcductasc h:ts to abolish noradrenalinc flunrescencc neuronal processes [.3X]. conrcrsion of phen$alanine to tyrosine in rat liver [4OJ. This ohservalion fitrthcr supports the hypothesis lhat availability of reduced pterin is a rcguintor~ factor in such hydro~yi~tti[~f~ reactions. Chcmicai‘sym~~~tlhectomy \+ith 6-l~ydro~iydnpatnine has been reportcd to have no efTcct on the levels of rat brain ptcridine rcctt~ctase [411 which would suggest that only ;I small proportion (probably less than IO per cent) of the total ptcridine reductase in brain is located in c~ttecholaminergic nwrons. Sonic will of course he present in serotonorgic neurons. but it would appear that the majority of ptcridinc reductase acti\Cty is cutraneuronal and tn;ty serve in, as yet. unidentified hydroxy&ttion reactions, Less is known about the control of serotonin synthesis in brain, although tryptophan hydroxylation is thought to be the most likely Gte for regulation to occur. Brain scrotonin levels arc also controlled by precursor availability and are increased by treatments that raise brain tryptophan le~ls [34]. Studies on the eiTects of ~idministered biopterin on serotonin sytlthesis have not heon reported. although it is likely that pterin levels are a regulatory &ctor tn the biosynthesis of the illdolc~i~~il~~s. by anaiog~ with the regukrtion of ~~tcch~~l~trnin~ biosynthesis, F~titii~.\ itrftl ,rctrt.sittt/iiit,/~itioti ~~~~~~~~~J~I.~. Several methyl trnnsferases are of ~~~l~~~~rt~tn~e in the mrtabolism of biogenic amines [42]. For example, both the hiosgnthesis of adrenaline and the inactivation of histamine involve n’-methylation rcactions, whereas the formation of melatonin and the inactivation of catecholamines involse O-methylation proccsses. A number of methylated derivatives of the hiogenic amines (e.g. mescaline and n;.?v’-dimcthyltryptaminc) produce psychotomimetic eifects in human beings and hence abnormal or excessive methylation of amine tlcurotransmitters has hccn impliuntcd in the xtiolopy of psychotic illnesses [itl]. This tr;ttlsmcth~l;ttion
Folxte
and pteridine
dcriwhes
hypothesis of s~t~~zophrcilia [44], originally proposed in 1952. is supported by the ability of the methyl donor m~~hionin~ to iniensify psychotic symptoms in a sign&canE proportion of schizophrei& patients. However. at present there is no comincing evidence for significant differences in the levels of psychotomimetic, methytated amines between schizophrenic patients and controls. Recent reviews have discussed the varying popularity of this transmethylation hypothesis [43.45]. A critical factor in the concept of an “endoeenous hattucinogen” would be the ability of mammalran tissues to synthesize psychotomimetic compounds such as dimethyttryptsmine. A significant advance was provided in 1961 by Axetrod [463 who demonstrated the presence, in rabbit lung. of an enzyme capable of forming ~~l~e~l~yt~~~~dtryptamine derivatives from the parent amine. This enzymic activity was also shown to be present in brain [47}, and N-methylated tryptamines have been reported to occur in various mammalian tissues [48], although the specificity of identification of these compounds using thin taycr chromatographic studies has been challenged [49J. In all the above work it had been tacitly assumed that the methyl donor in such ~ne~hylatjon reactions was S-adenosyl methionine (SAM). This assumption was challenged by Laduron [SO’J who proposed that CH,FH, may fLlnctioi1 directlv as methyl donor to biogenic amines. Previously ?H3FH, had merely been rcgardcd as the donor of a methyl group to homocysistcine in the biosynthesis of methionine [42]. Further reports from Laduron [51] and others [5X5?) described an cnpme from brain that could apparently donate the methyl group of CH,FH, to a variety of cttecholxnine derivatives to form both O- and N-methylated products. Characterization of this enzytne from brain showed it to be more active than SAM-de~~~ld~~lt amine methylases. and it was proposed that increasrd brain levels of CH,FH, may be responsible for the abnormal methytation processes thought to occur in schizophrenia 1541. A number of discrepancies among reported results have ted to a re-imcstigation of rhe specificity of this novel CH,FH,-dependent methylation reaction [55- 571. Lin and ~ar~~irnh~~hari [57] first noted that the amine reacrion products differed from those produced by SAM-dependent methylation processes and they suggested that the products formed were not due to direct methytation of the acceptor amines. The specificity of identification provided by gas ~~romatogr~~pily combined with mass spectrometry showed ihat the major product of the incubation of CH3FH4 and dopamine with enzyme extract was not N-methyt dopamine but 6,7-dihydro~yfetr~~hydro~soquit~~~t~r~e[SU]. C(~rr~spondingly, re~ral~ydro~~-~arboiines were formed from CH3FHd and tryptamine derivatives 159 -62). Such compounds are known to be formed by the non-enzymic condensation of formal~~ehy~~e with the parent amines. and the enzyme originally identified as a methyl transferax was subsequently shown to catatyse the release of formaldehyde from CH,FH, [63]. Current evidence suggests that this “formatdchydeforming activity” is identical with the generally distributed enzyme of one-carbon metabolism. methylene-FH, reductase 155, 561, although homogeneous
in tl~~r~tr~nsrnitt~r
mctaholism
IOfi
pre~ra~ions of the enzyme wtll be required to confirm this suggestion. The major physiological function of rne~hyleIle_~~~~ reductase is presumed to be the synthesis of CH,FH, (see Pearson and Turner [55] for discussionf, and the central role of this enzyme in providing methyl groups for subsequent synrhesis of SAM is indicated by the regulation of its activity by SAM levels 1551. However. in the presence of a suitable oxidant in r+lro a revcrsat of the methylene-FH, reductase reaction can occur which results in the conversion of CH,FH., to FH, and formaidehyde. The formaldehyde produced can then condense non-enzymicatty with a suitable acceptor such as a catechotamine or indoteamine. Since the amine does not enter directly into the enzymic reaction, previously quoted kinetic constants for amine substrates [Sl. 521 are of little significance. The analogous forma~i~~n of tetrah~droisoqt~~n~~linc ~~lk~l~~idsby the non-enzymic condensation of catechotamines with acetatdehydc or biogenic aldehydes has been reported to occur irr riro in mammalian tissues [64]. Such atkaloids have been implicated in the process of alcohol addiction and in some of the side-effects of DOPA therapy in Parkinson’s disease (for discussion see ref. 64). It is therefore feasible that form~~ldebyde-derived tetrahy-droisoyrtinofine and tetrahydr,-ji-carbrttine alkaloids may form itr rim in suffxcient concei~~r~lt~ons to modify the uptake or lil~tabolisrn of n~urotr~~ns* mittcrb. These alkaloids could arise by the forn~a~ion of “adive fortllatdehyde” from CH3FH4 through the action of Inethylene-Fan reductasc or from other formaldehyd~-generatilrg systems such as sarcosine dehydrogenax [I J. Recent s~udics by Ordonez and Vitlaroel C65] have indicated that the reversal of the methvlene-FH, reductase reaction may occur under certam conditions and these studies suggest that atkatoid formation in brain merits flirther investigarion. The incorrect idellti~cation of a number of products of one-carbon transfer reactions ES@-531 has indicated that any discussion of a rote for mcthytated amines in schizophrenia based sotetv on thin layer chromatographic studies for identif&g these compounds should be treated with caution. Future work should rely on more specific methods of product characterization, for example mass fr~~n~ent~~~raphy. Such an approach has recently been used to show the presence of amine-N-methylase activity in lung using either SAM or CH,FH, as methyl donor [66]. Therefore. although the majority of the activity originally identified as a fotate-dependent amine N-methyl transferase appears to bc due to the action of methytene FH, reductase, the presence of small amounts of a genuine f~?ta~~-d~pend~nt methylase cannot be precluded. I;cittrrt~ grail ~~~~~~~~~~~~ ~??~~~~~~~~~~.s~}I. At though a large body of evidence supports a role for glycinc as an it~hibitory neuro~rallsmitter. p~~r~i~ularly in the spinal cord [67]. our present knowledge of the metabolism of glycine in the nervous system is limited. However. both the synthesis and degradation of glycine are known to invofve fotate coenrymes 1671 and they represent further points of interaction between folates and neurotranstnitter metabolism. Neuronal gtycine is probably provided from serine through the action of serine t~ydroxymethyttransferase rather than by transport from the blood [6X]. Indeed.
the regional distribution of gXycine within the rat central nervous system correlates welt with the distribotion of serine hydroxymethvttransferase [141. The reversible reaction fatalysed by this err2yme invofves the traak of the 3-carbon atom of ~erine to FE%+: Serine + FH, -2 giycific + ~~~ethy~ene-~~~. An ~~ter~t~~e pathway to gIycine exists from giyoxyiate a~t~~o~~~~ rhe ~o~~r~but~on of this route has been estimated to be less significant than tha% from serme f6YjCSerine its& can be derived from glucose via 3-~h~~s~ho~~y~~rateor gfycerate with the former route predomjn~~t~~~ in brain irt riro [:70f. Ctycine catabolism may follow several different pathways [69]. Since the serine hydroxymethyltransferase reaction is readily reversible, glycine may be converted to serine in the presence of excess methylene-FEI, a~t~o~~l~ the presence of amines or other compounds Chat cm sequester the active formaldehyde moiety may regulate this reaction. An alternative route for the removal of glycine is through its oxidation to glyoxylatc by L-amino acid oxidase. an enzyme that is known to occur in brain 1717. However. the major mechanism for glycine catabolism, at least in peripheral tissues. is by the glycine cleavage system 1691 which catalyses the fofate-dependent d~~~box~~at~~~flof gtycine according to the following equation: GIycine -I FH, + NAIL?’ tiCC&
-t NH: -5 Me~~~~e~e-F~~ + NADH,
The reversible &cine cleavage system in rat Iiver is a rn~~~t~-e~?zyrn~ compIex i~~~o~~~~n~ four protein components f72f, which may be closcIy finked to serine hydroxymet~yttransferase. Little ts known of the properties of the g&&e cleavage system in brain tissue except that it is located Intra-rrtitochondriaiiy. Both se&e hydroxymethyitransf~rase and the gEy?xine cleavage system provide a means for the intrarn~tochQndrj~~ ~e~~~r~t~o~l of methylene FH4. Conversion of this folate derivative to CH,FN, is catalysed by a cytosolic enzyme and requires the transport of methylene F’H, across the inner lni&ochondrial membrane. which may h~olve a specific shuttle system for folates its ~~~~r~ti~~~~ above (171. There is at present little availab~c evidence concerning the compartmentation of f&c-dependent enzymes either in distinct cell types or specific regions of brain which may relate to the role of plycine as an inhibitory neurotransmitter. ~)~~p~~~~~r~~~~~.s j211’srtr(lics on ~nrcll ilh~. The recent interest in the ~~~~t~lb~~lis~~~ of brain folates is a consequence of the mcreasing clinical USC of Hood fota~e measurements, which has revealed that a number of neurological syndromes may be a result of folate deficiency {for discussion. see ref. 3). For example, severa! of the i&urn errors of &Sate mctabotism that lxwe been described show aspects of neurological ahnormalitp [XJ. Of particular interest with regard to the transrn~~by~at~o~ by~ot~es~s of schizophrenia is the description of 8 patient with rnethylene FH, reductase det%%Icp who ~xh~b~~e~ s~h~zo~hren~~ symptams that were refieved on treatment with f&c acid f733. The psychotic sy.mproms returned ~&en the fofate therapy was ~ij~~~~~i~l~~~. ESnr%Tuer. the signi%
Folate and pteridine derivatives in nemotransmitter
folates, pteridines and neurotransmitter metabolism. A number of such applications have been reported in the literature but these could usefully be extended. For example, a specific inhibitor of pteridine reductase has been used to investigate the significance of this enzyme to catecholamine biosynthesis [38]. The anti-~rkinsonian drug DOPA can be used to modifiy brain met~ionine levels and has been applied to demonstrate that the tie ww synthesis of methyl groups occurs to a significant extent in brain rather Fhan^ by transport of pre-formed methionine from blood [84]. Anti-folate drugs such as methotrexate inhibit a number of folate-dependent enzymes although with a lower affinity than that for dihydrofolate reductase. Drugs of this general type might, however. prove useful in studies of brain folate metabolism since dihydrofolate reductase is absent from this tissue. ~c~~~~v~~~~~~~~nr--~ shouid like to thank i\ndrew son for a helpful discussion of the manuscript.
Pear-
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535
(19%).
J. 5. Freeman and D. 1. Jenden. f-ifk SC+. 19, 949 (1Y7h). M. c-3. N. Boumt‘, S. K. Sharm;rn ilnd R. li. Snector,
OF FOLATE AND PTERIDINE DERIVATIVES NEUROTRANSMITTER METABOLISM
IN
ANTHONYJ. TURNER Department of Biochemistry, University of Leeds, 9 Hyde Terrace, Leeds LSZ 9LS. England The biologically active folate coenzymes are the derivatives of t~trahydrofolic acid (FH4) that function in the transfer of l-carbon groups in a variety of reactions in purine, pyrimidine and amino acid metabolism [I]. The ptcridine moiety, in addition to forming an integral part of the folic acid molecule. is also required as a donor of reducing equivalents in several amino acid hydroxylation reactions. A number of defects in both folate and pteridine metabolism are known to affect neurological function, suggesting an essential role for these compounds in the maturation of the nervous system and in synaptic dynamics [Z-4]. This review will concentrate on aspects of the metabolism of these compounds of current relevance to neuro~he~nistry and, in particLllar, to the metabolism of amino acid and amine ne~lrotransmitters. Such studies have been timited in the past because of the low concentrations of folatc derivatives in tissues. their instability and problems associated with their analysis. ~~~t~loil~~l r~~dioirnrn~~o~say procedures are now availabic for foiate measurements. The structures of folates and pteridines and the biochemistry of onecarbon transfer reactions have been described in detail elsewhere [I], although pathways relevant to this essay are shown in Fig. 1. Throughout this article the term ‘pterin’ will be used to refer to derivatives of the naturally occurring 2-am~~to-4-fiydroxypter~dine. as suggested elsewhere [I]. The role of folates in nucleic acid l~etabolis~l in brain will not be discussed here. Folatr ~?~~f~~~~~isi?l irr bruin. Although the synthesis of the pteridine ring system has been extensively studied in bacterial and insect systems. there is little information available concerning the biosynthesis of pterins in mammalian systems. However, it has been established that various mammalian tissues, including brain, possess the capacity to synthesize biopterin from GTP [S], and neurobiastoma cell lines have been shown to effect a similar synthesis [6]. The pathway of rnarnrn~~ljan pterin synthesis is presumed to be similar to that found in microbial systems. In contrast nialn~lal~a~ tissues do not have the capacity to synthesize folate derivatives and for human beings there is a daily dietary requirement of about 5Opg folic acid [Z]. The folic acid molecule is composed of a pterin linked through a ~?-amilloben~oyl moiety to one or more glutamate residues in T-carboxyl linkage (folate mono- and oligo-glutamates). 5-Methyltetrahydrofolate (CH,FH,) is the major form of folate present in serum and peripheral tissues BSwell as in the developing brain. However this is apparently not the case in the mature brain where almost SO per cent of the
total folate is present as FHJ oligoglutamat~ [4,7]. Folate levels in brain and cerebrospinal fluid are 2--3 times higher than those found in serum even in states of folate deficiency [S]. This indicates the importance of these compounds to the brain and suggests the existence of a regulatory mechanism for maintaining brain foiate levels. CH3FHa exists predominantly in a protein bound form in blood [9] and is concentrated into brain by a saturable uptake mechanism specific for folates [IO]. This requirement for the transport of reduced folates from blood is necessitated by the absence in brain of significant quantities of dihydrofolate rcductase [l If. Form~minotransfer~e, an enzyme which can contribute to the one-carbon pool in liver is also apparently absent from brain [12], suggesting that serine is the major endogenous source of one-carbon units in this tissue through the action of serine l~ydroxymetbyitransferase. Ail the enzymes required for the & IIOLW synthesis of ~-adenosy~methionine from serine are known to occur in brain (see Fig. 1). A number of studies [I2 -161 have examined the subcellular distributions of folate-metabolizing enzymes in nerve tissue and although there is general agreement on the cytosolic location of methylene FE& reductase, rn~t~ionin~ synthetase and methionine adenosyi transferase, discrepancies are apparent in the reported distribution of serine bydroxymethyitraI~sfer= ase. Davies and Johnston [13] and McClain et si~nificallt proportions &. [12] have reported (20-30*,,) of serine hydroxymethyltransferase in the FH4
ItGlycine
Methyl&FH,
4
Serine
HCHU
X
11’ CH, FH4 2
Homocysteine
j
A-W
w 4-
PPP;
-IS-Adenosyl
methibnine
Fig. 1. ~~t~rc~r~~~rsi~n of FH, derivatives in relation to amino acid and amine metabolism. The principal enzymes involved are: (1) merhylene-FH, reductase (EC 1.1.1.68); (2) CHIFHl homocysteine methyltransferase (EC 2.1.1.13); (3) methionine sdeaosyltransferase (EC X.1.6); (4) serine hydroxymethyltransferase (EC 2.1.2.1). The association of FH, with formaldehyde is presumed to be non-enzymk
cytosol. wilh the majority of lhe remaining activity occurring in the mitochoncirial fraction as in liver. In contrast. other workers hate conoi~idcct tktt this enzyme occurs almost cxclitsively in tnit~~~ll(~l~~~ri~ with negligible activity being present in the cytosol [14 Ih]. The dis~rcp~tt~~ies may reflect the d&rences in ~~o~~o~~~~is~tti~~~~ and fr~t~ti~~n~ttiot~conditions emplo>cd in these studies. A careful re-il~~~sti~~ti[~n of the l~~~~tlis~~ti~)tlof serinc hytfroxymetl~yltransfer3se in brain is inlp[~rt~ttl~ because of its possible involvement in a one-cnrbon shuttle system across the inner tnitochondrial mcmhrane [ 173. which would require both cytosolio and mitochondrinl forms of the emyme. Although a considerable proportion of cellular folates are present 21s their olipoglutamate derivatives. most studies on folate-metaboliziiig enzymes have tended to use the monoglutamates as substrates because of their ready ~tv~ii~tbility. However the oli~~~~lut~rn~~e forms of the f&tcs have been shown to be better substrates than the n7onoglLttalTintes for 21 number of folatc_iiitercctnvLrtit~~ enzymes [ 18 Xl]. Thus the oiigoplutamnte derivatives may not only represent storage forms or the folatcs as prcviottsly suggested [I]. but tnay function both as natural substratcS and as regulators of intr~~~ellul~tr folate-dependent reactions. Such possibilities should bt: borne in mind in studies on the enzymology of folate motabolistn. 7-/W lolc f!!’ ptefYrVi irr the S\‘lifhL’Si.\ ‘ji hic&gi’ltic* ~tt~ti~s. nteprtmary and rate-limiting step tn the svnthesis of the catecholamines involves the convct&n I..-tyrosi no r-?.4-dihydrosSiphcnyl3latiille of to (DOPA) by the myne tyrosine hydrosqlasc [Zl]. This enzyme catalyses a 3-substrate reaction involving tyrosine, oxygen and 3 reduced plerin cokictor. (probably telrah?idrohiopterin, B&i as shown in reaction (1): I.-tyrosinc
+ BH,
-C 0, ----)DOPA
BH,
+ NADH
+ BH,
“r H” ---;‘BH,
+- HI0
+ NAD
(1)
(2)
Regeneration of the reduced ptcrin is carried out by a pyridinc nucleotidc-depelidcnt enzyme. dihydropteridine reductase [12] (reaction 2). Phenyialaninc [3] and tryptophan hydroxytascs [1?4] also use ptcrin cofactors for the donation of reducing equivalents and similarly require pteridinc reductasc for regenerating the reduced cofactor. The related enzyme dtliydr~~folate rcductasc prob~tbl~ plays no role in the supply of Rtt, [25]. The re~Lt~~ttioi~ of ~tte~l~~~l~~rninesynthesis occurs mainly at the tyrosine hydroxylase step and may involve long-term modifi~~~io~~s of the amount of tyrosinc hydroxylase protein (tr~tns-syn~ptic indu~ti(~n) ;Ind short-term regulatory proccsses independent of changes in enzyme levels [Xl. This review will consider only the latter mechanisms, It is now well cstahlished that dopamine and noradrenaline inhibit tyrosine hydroxylase competitively with respect to the ptcrin cofactor [I??). Such ohservations suggest that release of catecholatnines from the nerve ending may result in an increase in the rate of amine synthesis due to the relief of end-product inliibi~ion of tyrosinc hydroxylase. Indeed, several studies have dernl~lis~~~ted an increase in the rate of
The synthesis of a number ol’ ncurotratismittcr~ including the catccholamincs may he regulated by prccursor availability (for disc&on see ref. 14). For example, elaation of tyrosine levels can bring about a transient incrensc in catccholaniinc synthesis [3S] and it has also been proposed that the levels of reduced ptcrin cofactor may regulate tJrosinc hydro~yl~ttion and amine synthesis [IS]. This suggestion is s~st~tin~d by the ~~erno~i~~r~iti~~rithat ir~tr~~~cli~ri~ula irtjection of bi~~pteriI1 [3’7j or its addition to sympathetic nerve tissue in culture [3X] or ~~n~tpt(~~~~t~~~l preparations [39] rcsitlls in ai increoscd conversion tyrosine to catecholnmincs. of an inhibitor of pteridinc rcductasc h:ts to abolish noradrenalinc flunrescencc neuronal processes [.3X]. conrcrsion of phen$alanine to tyrosine in rat liver [4OJ. This ohservalion fitrthcr supports the hypothesis lhat availability of reduced pterin is a rcguintor~ factor in such hydro~yi~tti[~f~ reactions. Chcmicai‘sym~~~tlhectomy \+ith 6-l~ydro~iydnpatnine has been reportcd to have no efTcct on the levels of rat brain ptcridine rcctt~ctase [411 which would suggest that only ;I small proportion (probably less than IO per cent) of the total ptcridine reductase in brain is located in c~ttecholaminergic nwrons. Sonic will of course he present in serotonorgic neurons. but it would appear that the majority of ptcridinc reductase acti\Cty is cutraneuronal and tn;ty serve in, as yet. unidentified hydroxy&ttion reactions, Less is known about the control of serotonin synthesis in brain, although tryptophan hydroxylation is thought to be the most likely Gte for regulation to occur. Brain scrotonin levels arc also controlled by precursor availability and are increased by treatments that raise brain tryptophan le~ls [34]. Studies on the eiTects of ~idministered biopterin on serotonin sytlthesis have not heon reported. although it is likely that pterin levels are a regulatory &ctor tn the biosynthesis of the illdolc~i~~il~~s. by anaiog~ with the regukrtion of ~~tcch~~l~trnin~ biosynthesis, F~titii~.\ itrftl ,rctrt.sittt/iiit,/~itioti ~~~~~~~~~J~I.~. Several methyl trnnsferases are of ~~~l~~~~rt~tn~e in the mrtabolism of biogenic amines [42]. For example, both the hiosgnthesis of adrenaline and the inactivation of histamine involve n’-methylation rcactions, whereas the formation of melatonin and the inactivation of catecholamines involse O-methylation proccsses. A number of methylated derivatives of the hiogenic amines (e.g. mescaline and n;.?v’-dimcthyltryptaminc) produce psychotomimetic eifects in human beings and hence abnormal or excessive methylation of amine tlcurotransmitters has hccn impliuntcd in the xtiolopy of psychotic illnesses [itl]. This tr;ttlsmcth~l;ttion
Folxte
and pteridine
dcriwhes
hypothesis of s~t~~zophrcilia [44], originally proposed in 1952. is supported by the ability of the methyl donor m~~hionin~ to iniensify psychotic symptoms in a sign&canE proportion of schizophrei& patients. However. at present there is no comincing evidence for significant differences in the levels of psychotomimetic, methytated amines between schizophrenic patients and controls. Recent reviews have discussed the varying popularity of this transmethylation hypothesis [43.45]. A critical factor in the concept of an “endoeenous hattucinogen” would be the ability of mammalran tissues to synthesize psychotomimetic compounds such as dimethyttryptsmine. A significant advance was provided in 1961 by Axetrod [463 who demonstrated the presence, in rabbit lung. of an enzyme capable of forming ~~l~e~l~yt~~~~dtryptamine derivatives from the parent amine. This enzymic activity was also shown to be present in brain [47}, and N-methylated tryptamines have been reported to occur in various mammalian tissues [48], although the specificity of identification of these compounds using thin taycr chromatographic studies has been challenged [49J. In all the above work it had been tacitly assumed that the methyl donor in such ~ne~hylatjon reactions was S-adenosyl methionine (SAM). This assumption was challenged by Laduron [SO’J who proposed that CH,FH, may fLlnctioi1 directlv as methyl donor to biogenic amines. Previously ?H3FH, had merely been rcgardcd as the donor of a methyl group to homocysistcine in the biosynthesis of methionine [42]. Further reports from Laduron [51] and others [5X5?) described an cnpme from brain that could apparently donate the methyl group of CH,FH, to a variety of cttecholxnine derivatives to form both O- and N-methylated products. Characterization of this enzytne from brain showed it to be more active than SAM-de~~~ld~~lt amine methylases. and it was proposed that increasrd brain levels of CH,FH, may be responsible for the abnormal methytation processes thought to occur in schizophrenia 1541. A number of discrepancies among reported results have ted to a re-imcstigation of rhe specificity of this novel CH,FH,-dependent methylation reaction [55- 571. Lin and ~ar~~irnh~~hari [57] first noted that the amine reacrion products differed from those produced by SAM-dependent methylation processes and they suggested that the products formed were not due to direct methytation of the acceptor amines. The specificity of identification provided by gas ~~romatogr~~pily combined with mass spectrometry showed ihat the major product of the incubation of CH3FH4 and dopamine with enzyme extract was not N-methyt dopamine but 6,7-dihydro~yfetr~~hydro~soquit~~~t~r~e[SU]. C(~rr~spondingly, re~ral~ydro~~-~arboiines were formed from CH3FHd and tryptamine derivatives 159 -62). Such compounds are known to be formed by the non-enzymic condensation of formal~~ehy~~e with the parent amines. and the enzyme originally identified as a methyl transferax was subsequently shown to catatyse the release of formaldehyde from CH,FH, [63]. Current evidence suggests that this “formatdchydeforming activity” is identical with the generally distributed enzyme of one-carbon metabolism. methylene-FH, reductase 155, 561, although homogeneous
in tl~~r~tr~nsrnitt~r
mctaholism
IOfi
pre~ra~ions of the enzyme wtll be required to confirm this suggestion. The major physiological function of rne~hyleIle_~~~~ reductase is presumed to be the synthesis of CH,FH, (see Pearson and Turner [55] for discussionf, and the central role of this enzyme in providing methyl groups for subsequent synrhesis of SAM is indicated by the regulation of its activity by SAM levels 1551. However. in the presence of a suitable oxidant in r+lro a revcrsat of the methylene-FH, reductase reaction can occur which results in the conversion of CH,FH., to FH, and formaidehyde. The formaldehyde produced can then condense non-enzymicatty with a suitable acceptor such as a catechotamine or indoteamine. Since the amine does not enter directly into the enzymic reaction, previously quoted kinetic constants for amine substrates [Sl. 521 are of little significance. The analogous forma~i~~n of tetrah~droisoqt~~n~~linc ~~lk~l~~idsby the non-enzymic condensation of catechotamines with acetatdehydc or biogenic aldehydes has been reported to occur irr riro in mammalian tissues [64]. Such atkaloids have been implicated in the process of alcohol addiction and in some of the side-effects of DOPA therapy in Parkinson’s disease (for discussion see ref. 64). It is therefore feasible that form~~ldebyde-derived tetrahy-droisoyrtinofine and tetrahydr,-ji-carbrttine alkaloids may form itr rim in suffxcient concei~~r~lt~ons to modify the uptake or lil~tabolisrn of n~urotr~~ns* mittcrb. These alkaloids could arise by the forn~a~ion of “adive fortllatdehyde” from CH3FH4 through the action of Inethylene-Fan reductasc or from other formaldehyd~-generatilrg systems such as sarcosine dehydrogenax [I J. Recent s~udics by Ordonez and Vitlaroel C65] have indicated that the reversal of the methvlene-FH, reductase reaction may occur under certam conditions and these studies suggest that atkatoid formation in brain merits flirther investigarion. The incorrect idellti~cation of a number of products of one-carbon transfer reactions ES@-531 has indicated that any discussion of a rote for mcthytated amines in schizophrenia based sotetv on thin layer chromatographic studies for identif&g these compounds should be treated with caution. Future work should rely on more specific methods of product characterization, for example mass fr~~n~ent~~~raphy. Such an approach has recently been used to show the presence of amine-N-methylase activity in lung using either SAM or CH,FH, as methyl donor [66]. Therefore. although the majority of the activity originally identified as a fotate-dependent amine N-methyl transferase appears to bc due to the action of methytene FH, reductase, the presence of small amounts of a genuine f~?ta~~-d~pend~nt methylase cannot be precluded. I;cittrrt~ grail ~~~~~~~~~~~~ ~??~~~~~~~~~~.s~}I. At though a large body of evidence supports a role for glycinc as an it~hibitory neuro~rallsmitter. p~~r~i~ularly in the spinal cord [67]. our present knowledge of the metabolism of glycine in the nervous system is limited. However. both the synthesis and degradation of glycine are known to invofve fotate coenrymes 1671 and they represent further points of interaction between folates and neurotranstnitter metabolism. Neuronal gtycine is probably provided from serine through the action of serine t~ydroxymethyttransferase rather than by transport from the blood [6X]. Indeed.
the regional distribution of gXycine within the rat central nervous system correlates welt with the distribotion of serine hydroxymethvttransferase [141. The reversible reaction fatalysed by this err2yme invofves the traak of the 3-carbon atom of ~erine to FE%+: Serine + FH, -2 giycific + ~~~ethy~ene-~~~. An ~~ter~t~~e pathway to gIycine exists from giyoxyiate a~t~~o~~~~ rhe ~o~~r~but~on of this route has been estimated to be less significant than tha% from serme f6YjCSerine its& can be derived from glucose via 3-~h~~s~ho~~y~~rateor gfycerate with the former route predomjn~~t~~~ in brain irt riro [:70f. Ctycine catabolism may follow several different pathways [69]. Since the serine hydroxymethyltransferase reaction is readily reversible, glycine may be converted to serine in the presence of excess methylene-FEI, a~t~o~~l~ the presence of amines or other compounds Chat cm sequester the active formaldehyde moiety may regulate this reaction. An alternative route for the removal of glycine is through its oxidation to glyoxylatc by L-amino acid oxidase. an enzyme that is known to occur in brain 1717. However. the major mechanism for glycine catabolism, at least in peripheral tissues. is by the glycine cleavage system 1691 which catalyses the fofate-dependent d~~~box~~at~~~flof gtycine according to the following equation: GIycine -I FH, + NAIL?’ tiCC&
-t NH: -5 Me~~~~e~e-F~~ + NADH,
The reversible &cine cleavage system in rat Iiver is a rn~~~t~-e~?zyrn~ compIex i~~~o~~~~n~ four protein components f72f, which may be closcIy finked to serine hydroxymet~yttransferase. Little ts known of the properties of the g&&e cleavage system in brain tissue except that it is located Intra-rrtitochondriaiiy. Both se&e hydroxymethyitransf~rase and the gEy?xine cleavage system provide a means for the intrarn~tochQndrj~~ ~e~~~r~t~o~l of methylene FH4. Conversion of this folate derivative to CH,FN, is catalysed by a cytosolic enzyme and requires the transport of methylene F’H, across the inner lni&ochondrial membrane. which may h~olve a specific shuttle system for folates its ~~~~r~ti~~~~ above (171. There is at present little availab~c evidence concerning the compartmentation of f&c-dependent enzymes either in distinct cell types or specific regions of brain which may relate to the role of plycine as an inhibitory neurotransmitter. ~)~~p~~~~~r~~~~~.s j211’srtr(lics on ~nrcll ilh~. The recent interest in the ~~~~t~lb~~lis~~~ of brain folates is a consequence of the mcreasing clinical USC of Hood fota~e measurements, which has revealed that a number of neurological syndromes may be a result of folate deficiency {for discussion. see ref. 3). For example, severa! of the i&urn errors of &Sate mctabotism that lxwe been described show aspects of neurological ahnormalitp [XJ. Of particular interest with regard to the transrn~~by~at~o~ by~ot~es~s of schizophrenia is the description of 8 patient with rnethylene FH, reductase det%%Icp who ~xh~b~~e~ s~h~zo~hren~~ symptams that were refieved on treatment with f&c acid f733. The psychotic sy.mproms returned ~&en the fofate therapy was ~ij~~~~~i~l~~~. ESnr%Tuer. the signi%
Folate and pteridine derivatives in nemotransmitter
folates, pteridines and neurotransmitter metabolism. A number of such applications have been reported in the literature but these could usefully be extended. For example, a specific inhibitor of pteridine reductase has been used to investigate the significance of this enzyme to catecholamine biosynthesis [38]. The anti-~rkinsonian drug DOPA can be used to modifiy brain met~ionine levels and has been applied to demonstrate that the tie ww synthesis of methyl groups occurs to a significant extent in brain rather Fhan^ by transport of pre-formed methionine from blood [84]. Anti-folate drugs such as methotrexate inhibit a number of folate-dependent enzymes although with a lower affinity than that for dihydrofolate reductase. Drugs of this general type might, however. prove useful in studies of brain folate metabolism since dihydrofolate reductase is absent from this tissue. ~c~~~~v~~~~~~~~nr--~ shouid like to thank i\ndrew son for a helpful discussion of the manuscript.
Pear-
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535
(19%).
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