Inhibition of tryptophan pyrrolase by serotonin, epinephrine and tryptophan analogs

ARCHIVES

OF

BIO(‘HEMISTRY

ANI)

BIOI’HYSI(‘S

Inhibition of Tryptophan Epinephrine and EARL

FRIEDEN,

G. WAYYE

176-182

92,

(1961)

Pyrrolase Tryptophan WESTMARK

From the Department of (‘henristr!], Florida Tallahassee, Florida Received

August

by Serotonin, Analogs’ il?;~ JOSEPH

M. SCHOR2

Stale I,nicersit!y,

1, 1960

Jlanl, tryptophan analogs were found to effectively block the conversion of tryptoto kynurenine in the tryptophan pyrrolase reaction. Side-chain variants surh as indole&acrylic acid were competitive inhibit,ors of trypt,ophan. Substitution of the indole ring produced several noncompetitive inhibitors, of which serotonin was t,he most effectjive. Epinephrinc and certain other phenols were also potent now competitive antagonist,s. The inhibition by epinephrine and other phenols may br due t,o interaction at, the beme sit,e for this enzyme. None of the inhibitors test,ed induced tr,vptophan pyrrolase in the ntlren:tlectolnizetl rat, when administered in phan

Since the discovery of trypt(ophan pyrrolase3 (1) as an inducible enzyme in rat’s by Knox and Mehler (t-‘), c*onsiderable work has appeared on the iti oioo effects of various substanceson t’his enzyme. It was found that in addit’ion to t,ryptophan, compounds lvhich produce a metabolic stress could also induce the forrnat’ion of trypfophun pyrrolase (3). In this way t,he role of the adrenal hormones in tryptophan pyrrolaxe format’ion was found by Thomson and l\likuta (4) and by Knox (5). Subsequently, Schor and Frieden (0) found that both insulin and nlloxan could also induce t,ryptophan pyrrolase production. However, only very limited studies on the in vitro effect of compounds on this enzyme have been reported (G-10). Some 1 This research was sllpported in part by Research Grant, C-23i5, from The National Cancer Institute, N:tt,ional Institutes of Health, 1:. S. Public Healt,h Service. 2 Present address: The Lederle Laboratories, Pearl River, Kew York. 3 The designation trypt,ophnn yyrrolase is preferred to the previously used name, tryptophan peroxidase. The reaction cat,alyzed is the c’onversion of tryptophan t,o kynurenine, probably via formylkynlwenine. 176

data on the in d-o inhibition of tryptophan pyrrolase was briefly reported in our earlier paper (6). In t’his paper, we report, the inhibition of t,ryptophan pyrrolase by serotonin, tryptophan analogs, indole derivatives, and some phenols.

Trgptophan pgrrolase :tct,ivity wxs determined as described earlier iti), a method essent.ially simlar t,o that of Knox (5). When compollnds were tested in aivo they were injected 6 hr. before the removal of the livers for tryptophan p>-rrolase analysis. In the in ailro experiments, tryptophan pyrrolase was induced 6 hr. earlier by :IIL injertion of 1.0 g./kg. L-tryptophen. The livers were removed and a 5’;; homogenate prepared. This homogenate served as a source of enzyme and was stable for at least 2 weeks svhen kept at, -20°C. R’hen a t rgptophan analog \vvas used, a blank with t,he analog was subtraot,ed from the values obtained with 3 X 1O-5 JI tryptophan and analog. A tissue control wts also subtracted. The tryptophnn pyrrolase activity was determined over a 3.hr. incubation period at 37°C. during which time the rate was subst,antially linear. The source of unusual analogs is given in Table I. In Tables 1 and II, the data have been expressed as per cent inhibition in the presence of the indicated concentration of the tryptophan analog when compared to the t ryptophan control.

TItYPTOPHdX TABLE

PYHHOL.kHE

Inhibitor

Molarity

inhibition~~h

-

Indole-3.:tcet,ic acid lndole-3-propiorlic acid Indole-Zlmtyric acid Indole-3-acrylic acid Indole I,-Tryptophan 8-Meth~ltr~-ptophatl (isomer r\ )( @-Met hylt rypt>ophan (isomer 13)r Trypt”“‘~” ‘I’rpptamine 5.H~dros~tr~ptoph:IIl” Serotonin 5-F-‘l’r~pt~o~,har~~’ 6.F-Tr!ptophan E 5.Mct,hylt ryptophan B-?uletIl\ltr~ptophnll c 5-H~dros~illdole-:~arctic acid 5~Meth~liridole-2-r:irhosylic acid

1 69 51 34 43 , 52i $2

, 9X 87 0.012 i 9 36 ’ 16 1.6 32~ ~ Il.1 31~ 22

1.1

50, 38; 29 881 7“ 08’ / 95

n Thr experimental conditions are given in dein a prwious paper (6). I’ Ixw than 26’% inhibition at 3.0 1n3l was produwd 1)~ the follow\-ing compounds: ncetyl-I,t ryptophan. kynln-enic acid, imidazole, hippuric acid. llist:~lllirle,ph~,Il?lrner~:lptoacetic arid, 2,&di~hloroplrcllosv:tcetic acid, n:lphth:tlelieacetic, acid, 4.~hlorophprrory;tcctic x-id, 2-furylacrylic acid. pyritiosal, adenylic acid, glmnylic arid, proline, and hist idinc. c (kncrollsly provided by Dr. II. It. Snyder of the I)c~lxtrtmrnt, of Chemistry, Ilniversity of Illinois. il (;cnerously donated I),v I)nn Broida of the Sigma Cllrmical Co. All other compounds bvere purchased from thr Sigma Chemical Co. or the Nutritional Biochemirals Corp. Conlpo~nds were rerrystallizwl when necessary. F (knerously provided 1)~ l>r. I<. I>. Bergmann of I hc: Hchrcw t;nivcrsitT, Jerusalem. / ‘l’hr Kl’s for the side-chain variants were estinlated from the competitive inhibition equ:~tion. Thr KI’S for the substituted indole ring c‘ompounds lvere estimated from the noncompetit,ive inhibit ion equation. However, it was noted that an irregularity esistetl in that, some of the Kr’s appeared IO depend on the inhibitor ronrentrw tail

177

I

IXHI~~ITION OF TRI’I’WPHA~ PTRROL.~SE BY TRYPTOPAAN AXAL~GS Per cent

INHIRITIOS

tion. For example, the estimated Kl for indole-% acr?;lic acid at 12, 50, and 100 PM was 7, 12. and 16 ~d4, respectively. A similar variation in the same direction was noted in the estjim:tt,ed Kl’s ~OI tryptamine 311d serotonin. While it is I)elievcd that this fluctuation exceeds psperin~ent,al varintion, the crudeness of the rnzym~ system used makes it unprofital)le to probe this point further until the effcrt can be checked with n homogeneous enzyme. If the variation in Kr in also found with pure enzyme, then more complex inhibitory mechanisms nrust be sought, such as emphasized in the review of Hearon et ctl. (11). The inhibitor constant Kl , was the apl)ropriate graphs or from the equations given below. For competitive inhibition,

estimated from the solution of

so that in a 13 versus P/(S) plot, a romprtitive hibitor is indicated by an identical interrupt an increased negative slope. For nonrompetitivc inhibition,

2,=JL!-&1+

‘f! 11 I

“I

inI)rlt

1: II 1 (8)

where Kl = K~:JI Thus a Ilorlc*oml)etitivr irlhibitor gives a 1%versus V/(S) plot of lines of itlerltiral slope and reduced intercepts. In the above equations, the symbols r~spd hnvp the usual meanings as follows: (’ = velocity of the reaction in t,hr prrsenrc of inhibitor, at ronrentration (I). = maximrim vrlority at a constant rnz,vmc 1-m c’ollc’plrtr:Ltioti. Ii, = dissociation constant of the rrrzymp-illhibitor cwmples. kr~s~ = dirsociation ronstant of thr enzymes;rll)slratp-irlhit)it.or cornpIes. (n) = substrate ronrentr:ttion.

X survey of the effect of trypt,ophan analogs and other inhibitory compounds on tryptophan pyrrolase is summarized in Tables I and II. The percentage inhibition of the enzyme is given n-hen the tryptophan concentjration in the reaction mixture was fixed at 3.0 X lo-” M. Tryptophan pyrrolase is senskire to a wide variety of tryptophan analogs and derivatives and certain phenols. JIost of the inhibit’ors of this en-

178

FRIEDEN,

TABLE II OF TRYPT~PK~N BY PHENOLS

INHIBITION

Per cent Inhibitor

Molarity

Phenol C&echo1 Resorcinol Phloroglucinol Hydroquinone p-Quinone L-Dihydroxyphenylalanine Tyrosine Arterenol bitartrate 1,.Epinephrine cu++

WESTMARK

SCHOR

PYRROLASE V

inhibition0 Estimated KIb

-

838 69 931 i 78~ 81 68~ 99 99 93:

24 89 81 54 38 18 81~ 56 90 69 74

0.38m~ 24 0.032 16 0.20 0.46 0.075 0.043 0.04

41 20

42

I

46

26’ 22

84

M

( 51

99

71 97

~ 47 -

d -

3.2 0.13 0.037

y The experimental conditions are given in dein a previous paper (6). * Approximate KI’S are estimated from the noncompetitive inhibition equation. c The inhibitor was tested at 9.0 X lo-” J1 inst,ead of 1.0 X 1OW $1. d The inhibitor was tested at 5.0 X 1OV M instead of 3.0 X lo-” M. tail

AiXD

FIG. 1. Competitive and noncompetitive inhibition of tryptophan pyrrolase by four tryptophan analogs as shown by 2) versus v/(s) plots. For both axes the velocity is expressed as the increase in optical densit,y at 360 rnp in the 3-hr. incubation time. The unit of substrate concentration is millimolar.

acetic, -3-propionic, -3-butyric, and -3acrylic acids as list’ed in the last column of Table I are 0.91, 0.29, 0.16, and 0.012 mM, eyme are congeners of kyptophan \Cth four respect’ively. Though such constants, obmajor st,rucbural features: (a) variations in tained in a crude enzyme system, are not the alanine side chain; (b) substitutions in highly accurate or reliable, they do indicate the indole nucleus, particularly in t>he ben- t,he strong influence of side-chain variations zene ring; or (c) both of t’hese; and (d) on inhibitors of t)ryptophan pyrrolase. The two isomers of P-methyl tryptophan and epinephrine and certain phenols. n-trypt’ophan are considerably less effective SIDE-CHAIN VARIANTS OF TRYPTOPHAN as inhibitors, but indole produced a reducSide-chain variants of tryptophan are tion in rate comparable to indole-3-propionic listed in Table I. Several side-chain analogs acid, and t#heK, for tryptamine is comparable to indole-3-butyric acid.4 are effective ant,agonists of kypt,ophan pyrrolase with indole3-acrylic acid being SUBSTITUEXTS IN THE IXDOLE NUCLEUS t#hemost effective. As shown in Fig. 1, trypSubstitution on the indole ring produced t#amine and indole-3-acrylic acid are compet,itive inhibit,ors of tryptophan pyrrolase. a different, type of inhibit,or in t,he case of This is shown in a plot, of t’he data u versus 5-E’-tryptophan a,nd S-hydroxytrypt,ophan. In Fig. 1 t,he noncompetitive inhibition of U/(S) resultmingin lines wit’h a common intercept, V,, and an increased negat,ive slope. 1 Dashman and Feigelson (9) have reported Experimenk not included here indicate that t,hat indole-3.acetic, indole-3-propionic, indole-3indole-3-butyric acid is also a compet’itive butyric acids and G-mcthq-ltryptophaII,h~-ltr~ptophaI1 had simitype of antagonist. Thus all t,he side-chain lar inhibition constants to those reported in analogs studied were inhibitors of the com- Table I. Agreement was less close for inhibition petitive t,ype. Estimat’ed K1’s for indole-3- data for trypt>amine and indole.

179 these two compounds is indicated by lines of displaced inkrcept and parallel slopes in 1’ versus P/(S) plots. (i-F-tryptophsn is also as effectjive an antagonist, as the corresponding 5-substituted compound. Similarly, the equivalence of t#he 5 and 6 positions is indicated by the similar but lower level of ant,itryptophan activity, shown by the 5and CI-methyltryptophan. Another type of indole modification in tryptazan is also inhibitory. The st,rong inhibitory effect of 5-hydroxytryptophan on liver tryptophan pyrrolase was noted earlier by Ichihara et

h

1.0

SEROTONIN

al. (‘i).

In the next section, t,he strong noncompetitive inhibition of tryptophan pyrrolnse by serotonin is presented. The identical nature of the inhibition observed for serot,onin and Shydroxytryptophan may arise from the fact that’ during the :3-hr. inrubat,ion period used in these experiments, Shydroxytryptophan may be converted to serot,onin. A specific 5-hydroxytryptophan decarboxylase has been reported in liver and ot,her tissues (12). The ult,imate conversion of serotonin to 5-hydroxyindole+acetic acid via monoamine oxidase (13) is probably not, a significaut effect berause S-hydroxyindole-3-acetic acid is much lessinhibit,or.y.

The last several compounds in Table I represent, compounds with modifications in both t#heside chain and the indole nucleus. Scrotonin proved to be an extremely effective noncompetitive antagonist of trypt’ophan as shown in the upper graph of Fig. 2. The reversibilit,y of t)he serotonin inhibit’ion was est)ablished by an enzyme titration curve (14) asshown in t)he lower plot, of Fig. 2. It, would be of interest to be able to combinc t,hc skuctural alterations which proved to be most effective in blocking trypt,ophan pyrrolase wtivit8y. A compound such as 5-hydroxyindole-S-acrylic acid, not available at, present, combines t.he most, effective inhibitory fest)ures. However, for 5-hydroxyindole+acetic acid, the effects were in no way additive since t#hiscompound is a relatively poor inhibitor being considerably less effert)ivc than indole-s-acetic acid, indole, or any of the 5-hydroxy compounds.

ML.ENZYME FIG. 2. In the upper half of the figure, the now competitive nature of the serotonin inhibition is shown with the same coordinnt,es as in Fig. 1. Reversibilit,v of the srrotonin inhitGtion is indicated in the lower graph in :L 1’ versus enzyme concent,rnt,ion plot. The amount of enzyme is v:tricd t>>- using different volumes of 5’3 liver homogenate in 3.0 ml. reaction volume.

The import.ant contribut,ion of the S-hydroxy group suggested that the phenolic group might be an important feature of certain trypt’ophun pyrrolase inhibitors. It, has also been suggested that this enzyme is probably a heme prot,ein (15). Intjeract,ions of phenols and iron compounds are well known. The data on a number of phenols are summarized in Table II. Among the

180

FRIEDEK,

\VESTMARK

.8 EPINEPHRINE

0

.4

.8 “/S

ASD SCHOR

inhibition. The noncompetitive inhibition by phenols and polyphenols, such as epinephrine, may he readily pictured as involving imeractions at, t,he proposed hemeFe site in trypt’ophan pyrrolase. The comparable activity of the 5hydroxyindole derivat’ives might also he t,raced to phenolic interactions with t)he enzyme lie. However, a similar mechanism is not as readily acceptable for the ot’her 5- or (i-substituted t,rypt’ophans. It is also of int,erest t,o note that’ the presence of a st,rong electronegative group in the indole nucleus produces strong noncompet’itive inhibition. IN l'rvo EXPERIMEXTS OF TR~PTOPHAN

6 MLS.

ENZYME

3. soncompetit,ive inhibit,ion of trvptophan pvrrolase hy epinephrine. The coordinates are the same as in corresponding graphs in Figs. 1 and 2. In the lower graph in which the reversibility of the inhibition hy three phenols is tested, the symbols have the following identification: 0, no inhibitor; X, 3.0 X 10F5 ,%I epinephrine; 0, 3.0 X 1OP dl phenol; 0, 1.0 X lo-” :II hydroquinone. FIG.

more effective phenolic inhibitors are epinephrine, catechol, dihydroxyphenylalanine, and p-quinone. As shown in Pig. 3, the inhihition hy epinephrine was found t,o he noncompet,itive and reversible hy t’he method of Ackerman11 and Pot’ter (14). The inhihition by 1O-4~$1hydroyuinone (bottom curve in lower part of Pig. 3) is, however, pseudoirreversible. RELATION OF IXHIHITORS TO THE MECHAXISM OF ACTI~S 0F TR~PTOPHAK PYRROLASE

Conclusionsasto the active site and mechanism of an enzyme from purely kinetic dat#a are quite hazardous. Clearly, the Lalanine side chain is direct’ly involved in complex@,

with

enzyme,

since all variants

of the side chain produce only compet,itive

\\YTH IKHIBITORS PYRROLASE

Interest in tlryptophan pyrrolase inhihit,ors arose init~ially out of an interest, in the mechanism of t’he induced synt#hesisof this en zyme in rat liver. Monad (16) has observed t,he induction of the enzyme, fl-galact#osidase, by methylthiogalactoside, an inhihit’or of t’his enzyme. Therefore, t,he in vice effect of the st,rongestin rdro inhibitors was studied. The result,s are summarized in Table III. Small increases in tryptophan pyrrolase were ohserved in rerotonin and the 5-hydroxytrypt’ophan-injected adrenalectomixed rats, hut the effects were not’ yuantitatively comparable to the effects of tryptophan. It was not possible to test, serot,onin or 5-hydroxytryptophan at’ higher doses because of the toxicity of t,hese compounds. The effect, of indole-Ghutyric acid and indole-3-acrylic acid in normal Wistnr animals is probably a metabolic st’ressand is apparently due to the int~ervention of t,he adrenals, an effect noted earlier (4,5). Again, the effect, of any of these inhibitors did not approach that of tryptophan itself. Several lahoratories (10, 17)5 have rea-methyl-r)Lported that, wtryptophan, trypt,ophan and X-oc-acetyl-n-t’ryptophan showed a suhstrat#e-type induct,ion of liver t,rypt,ophan pyrrolase in adrenalectomized rats. These compounds show only a low order of in &o tryptophan pyrrolase inhihit,ion. It is also possihle t#hat# u-trypt,ophan

and N-a-acetyl-L-tryptophan

5 Also, I. Geschwind,

private

arc (bonrommrmication.

THYPToPH.%S

PYKHoL.ISE

TABLE ATTEMPTEI)

IN

Type

\'rvo

INDUCTION

OF

of ratCL

III

TRYI'TOPHAS

Compound

181

ISHIRITlC)S

PYRROLAS

WITH

injected

TRYPTOPIMN

Wistnr

None Tryptophan Indole-3.acrylic Indole-3.hutyric Rcrotonin adrenalect,omizedc

lu‘one Indole-3.acrylic Indole-3.but,gric Serotonin 5-H~drosytr~ptophnn

Micromoles kynurenine/ hr./g. liver

Dose lllg./llio

Wistar

ANALOGS

g. ml

-

1.53 13.0 1.01 4.6 3.41

200 200 100 25

acid acid

acid acid

75 50 15 75

acid

100

* + f f f

.54” 1.9 .58 2.4 .57

.x0 i .91 + 1.20 f 1.48 f 1.65 It

.24 .43 .35 .06 .20

3.07

SOIlC

lndole-3-nc>rylic

4.3

‘I ltach esperiment involved a group of 3-6 rats. Tryptophan pgrrolnse xtivit:; the indicakd (low. ‘) Standard deviation. r i\tlrell:Llrct,omizcd Wistnr rats respond with :L F~l0 fold inrrrase in enzyme phnn inject,ion. r( Sprague-I)awley rats tihow the usual proportionnl enqme incrrasr after The initial tryptophan pyrrolnse level is doubled.

Z:M

&

.i2

It

2.7

determined

6 hr.

after

vert,cd

t,o

hydrolysis

L-tBryptophan

in

Go

through

or transamination.

~MPLICATIOSS EPISEPHRISE

OF

SEItOTONlN

AND

IKHIRITIO;~

Aluch emphasis has been placed on the hormonal control of induced enzyme formation. That, tryptophan pyrrolase is under t~he in mko influence of a number of hormones including the adrenal cort,es and t,he pancreas is now well accepted (4-G). The experiments reported here also point to the possibility of at least an ilL do effect of t,wo hormones, epinephrine and serotonin. The difliculties of i~z Go administ,rat,ion of these compounds at the doses used did not permit a decisive conclusion as t)o their in Go c$fec%s. Xwareness of the metabolic importance of scrotouin and epinephrine has increased greatly during recent. years. While the neuml, pressor, and certain ot#her effects of these hormones are probably their most import,ant, biological roles, this work suggwts t,hat, t,heir effects on tryptophan met’abolism should not, be overlooked.

activity

aftrr

I.-trypt

L-tryptophxn

injcri

oion.

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\I-.

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('hC't,L.

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F..

in

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WESTMAKK

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ASD

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SCIIOR

TANAKA,

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J.

_