Alternate metabolic fate of 5-hydroxytryptophan

Vol.& No.4

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ALTERNATE METABOLIC Richard

OF 5-HYDROXYTRYPTOPHAN

FATE

P. Spencer*

Oct. 1960

and Norman

Zamcheck

the Gastrointestinal Research Laboratory, Mallory Institute of Pathology, the 2nd and 4th Medical (Harvard) Services, Boston City Hospital, the Department of Pathology, Boston University School of Medicine, and the Department of Nutrition, Harvard-School of Public Health. From

Supported C-209O(C6) Inc. Received

by Grants from the National Cancer Institute and C-4486 from the American Cancer Society,

September

26, 1960

The compound because

of its

production here is

conversion

by patients

evidence catalyzed

under

with

by enzymes

of

the

generated both

this

keto

from

tautomerases.

be produced

from

suggested

*Research

the

During

‘59).

ficity,

fate

also

metabolize

of this

pathway

We present of

5-HT which

tryptophan. is

unknown but

investigation.

equilibration Knox,

of interest excessive

tumors.

metabolic

significance

is

and its

carcinoid

which

The 2 indolylpyruvic

&

(5-HT)

to serotonin,

of an alternate

The biological is

5-hydroxytryptophan

Fellow,

acid

acid keto

(IPA)

and enol

investigations

tautomerases tautomers

catalyze

of IPA

of substrate

specifi-

(S-hydroxyindolyl-pyruvic

5-HT was noted Since

is

The Helen

fate

of

5-HT,

Hay Whitney 386

5-HIPA)

substrate

for

that

5-HIPA

can

(Sandler

et al,

'601,

distinct

from

evidence

5-HT by transamination a metabolic

acid,

to be a suitable

there

(Spencer

Foundation

its

Vol.3,No.4

BIOCHEMICAL

Oct. 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H NHe R&-COOH H OH TRANSAMINASE

R-C-C - COOH t ACTfC DEWLMOGENASE

I:

H OH R-b-I;-COOH

Figure

1.

Alternate

decarboxylative sented

that

5-HT with aminates

metabolic

conversion the hepatic

to serotonin.

is

likely

In addition,

of 5-hydroxyindolyllactic These metabolic

of 5-hydroxytryptophan.

enzyme involved

<-ketoglutarate tryptophan.

fate

acid

is pre-

in transamination the

data

(5-HILA)

interrelationships

Evidence

same one that

of trans-

indicating

production

from 5-HIPA

are given.

are shown in Figure

1.

EXPERIMENTAL Assays were based on the enol borate-tautomerase of Knox and Pitt

('571,

of IPA metabolism

(Spencer

HT was transaminated Civen's liver 60 pgm

('57)

method

with

centrifuged

pyridoxal

phosphate, buffer,

received

& Knox,

'59,

o( -ketoglutarate

(1 ml of

homogenate

0.57 M borate cuvette

which has been applied

supernatant at

105,000

Spencer,

method

to the study '60).

in borate

DL-Sby

from a 20% rat x g for

30 minutes,

and 100 )unoles of DL-5-HT,

pH 8.1).

20 )unoles of

in

The assay spectrophotometer d -ketoglutarate 387

to start

the

Vol.3,No.4

BIOChEMICAl

reaction.

Optical

328 mp, at which addition

of the

there

density point

d-ketoglutarate

the

resulting

Further

that

was J-HIPA

as 5-HIPA

4 mg L-amino

A spectral

tained

compound

merase

and Increase

the

oxldase,

5-HIPA

en01 tautomers. formation:

0.04

the

rate

gave an increase tryptophan

was

minutes.

5-HT 5-HT

shawn to have the Cuvettes

zero

borate

to the

resembled

The reaction present

that

ob-

was usually

to supply

of equilibration

of..the

tautoketo

and deteriorates

ethylenediamine

assay

and

after

tetraacetlc

acid

of degradation.

Transamination was zero order

from

as follows:

at time

is unstable

M dieodium

has been

1 rag catalase,

supernatant rate

the

IPA,

produced

scan of the product

liver

of 50 jnnoles

by transaminatlon.

5-HT added

rat

assay

this

'56).

and the product produced

near

appeared.

et al,

when 5-HT was transaminated.

performed.with

maximum

was provided

acid

and 0.5 pmoles

cuvette.

the

of time,

of tryptophan,

(Bentley

deaminated

same spectrum

of peak

After

at 328 my (and

in the

transaminatlon

evidence

was oxidatively

buffer,

O.D.

at

absorb.

the passage

peak with

same type

at 328 q

by transamination

0.17/10

a broad

from

ehcrwn to absorb

contained

in

Oct. 1960

were measured

does not

When 5-HT was replaced

of L-tryptophan, product

changes

, with

increase

scans revealed

wavelength).

(O.D.)

5-HT in borate

was a progressive

spectral

slows

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of 5-HT.

for

at least of

used

0.15

O.D,

in place

Fractionation

measured

by the

30 minutes. units/l0 of 5-HT, of rat 388

O.D.

A typical

minutes. the liver

at

328 v

reaction When L-

increase

was

eupernatant

by

Vol.3,No.4

solid

BIOCHEMICAL

awronlum

dialysis

sulfate

changed

ketoglutarate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(O-30%,

the

30-So%,

specific

activity

transaminase,

but

the

on L-tryptophan

and 5-HT

consistent

the

view that

transaminating

both

tryptophan

Sandler

'60,

based. on the

with

et al,

(l.l:l)

50% supernatant)

and

of tryptophan-o(

-

ratio

of the

remained the

Oct. 1960

activity

constant.

same enzyme

and 5-HT

This

is involved

(as suggested

adaptive

is

increase

in

by of the

enzyme) . That merases

5-HIPA

was a suitable

was shown as follows:

wa8 used to convert liver

in O.D.

was slow.

Upon

the

rate

increased.

for

a reversible

expected

constant

showed that the

rate

of a reversible addition

was

factor,

table

but

the

rendered

can utilize

5-HIPA

was tested

in the

system,

too

S-substitution

was

to the

dependent

found

does not

order

rate

rate).

in30 times.

supernatant,

the

actifor

of its

tautomerization Hence both

the

cocon-

activa-

IPA tautomerases

When DL-5-methyltryptophan oxidase

to be a suitable affect

order

upon glutathione

dependent)

acid

over

due to binding

ae substrate. Lyamino

first

first

of keto-enol

(non-cofactor

deviated

1 ml of supernatant

inactive

at a slower

and basal

it

(that

catalysis

(however,

Curves

tautomerizatlon

was added

IPA tautomerase

activity)

of

of keto-enol

When N-ethylmaleimlde vatable

system

The increase

from

Calculation

oxidase

(rat

slightly reaction.

acid

IPA tauto-

tautomerase

of supernatant,

creased

The L-amino

the

without

supernatant).

those

for

5-HT to 5-HIPA

addition

tinued

substrate

the 389

plus

tautomeraee substrate

tautomerases).

(hence Partially

Vol.3,No.4

BIOCHEMICAL

purified

preparations

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

IPA tautomerases

also

Oct. 1960

shaw the

same effects. Attempts oxidase

system

)unoles were

to convert by addition

DPNH (reduced initially

in O.D.

unsuccessful,

from

tryptophan

and neutralization,

and the

ization.

Synthetic

dehydrogenase 0.1).

IPA can also

and DPNH (in

at 340 m)z.

a nonspecific (Alivisatos for

the

summary,

o(-ketoglutarate strate production

for

borate

since

buffer),

and

Some comthe

upon deproteinwith

lactic

by a decrease

a distinct

process

ddrivative

lactic

acid

in

and not

to DPNH

dehydrogenase

was

reaction. 5-HT is

converted

transaminase. both

prevented

was removed

of an indole '60),

at 340 9.

be shown to react

was likely

addition et al,

required In

This

However,

dehydrogenase

system

agent

IPA

metaphosphoric

in O.D.

deamination

inhibiting

acid.

with

decrease

to convert

of lactic

by a decrease

oxidative

reaction,

cold

directly

by a minimal

failed

acid

and 0.74

nucleotide)

also

addition

L-amino

dehydrogenase

as measured

the

the

of the

in the

to indolyllactic in

DPNH was followed

lactic

The system

upon deproteinization

ponent

of

to 5-HILA

diphosphopyridine

at 340 mp.

produced

5-HIPA

of the

of 5-HILA

to 5-HIPA 5-HIPA

IPA tautomerases. from

5-HIPA

is cited.

390

by tryptophan-

is a suitable Evidence

subsuggesting

Vol.3, No.4

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Oct. 1960

REFERENCES Alivisatos, S.G.A.: Mourkides, G.A.; Jibril, A.: Nature 186: 718, 1960. Bentley, J.A.; Faraar, K.R.; Housley, S.; Smith, G.F.; Taylor, W.G. : Biochem. J. 64:440, 1956. Civen, M.: Thesis. Harvard University, 1957. Knox, W.E.: Pitt, B.M.: J. Biol. Chem. 225:675, 1957. Sandler, M.; Spector, R.G.; Ruthven, C.R.J.; Davison, A.N.: Biochem. J. 74:42P, 1960. Spencer, R.P.: Thesis. Harvard University, 1960. Spencer, R.P.; Knox, W.E.: Abstr. 136th Meet. Am. Chem. sot. p. 41c, 1959.

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