THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 256, No. 17, Issue of September 10, pp. 9167-9173, 1981
Printed in U.S.A.

Thyroid Hormone Synthesis in Thyroglobulin

THE MECHANISM OF THE COUPLING REACTION*

(Received for publication, February 13, 1981)

Jean-Michel Gavaret, Hans J. Cahnmann, and Jacques Nunez

From the Unite de Recherche sur la Glande Thyroide et la Regulation Hormonale (Institut National dela SantP et de la
Recherche Medicale), 94270 Bicgtre, France, andthe National Instituteof Arthritis, Diabetes, and Digestive and Kidney
Diseases, National Institutesof Health, Bethesda, Maryland 20205

~-14C]Tyrosine-labeled noniodinated hog thyroglob- product. In a subsequent investigation (9), it was proven by 2
ulin was iodinated enzymatically and nonenzymatically independent methods that the pyruvic acid arose from dehy-
(iodine, iodide-chloramine-T, pH 7.4, or iodine mono- droalanine residues in the iodinated thyroglobulin.
chloride, p.H 8.1). This led to similar levels of iodine Since thyroid hormone residues are synthesized in thyro-
incorporatzon as well as of thyroid hormone synthesis. globulin not only by enzymatic iodination (10-14), but also by
Iodine monochloride at pH 5.5 formed hormonogenic nonenzymatic iodination (15-18), the question arose to what
iodotyrosine residues, but no hormone residues. The degree the mechanisms of enzymatic and nonenzymatic cou-
latter were formed when the iodinated thyroglobulin plingdiffer. In order to approach this question, we have
was brought topH 8.6 and then treatedwith horserad- determined whether the products of the lost side chain are
ish peroxidase and glucose-glucose oxidase in the ab- the same or different depending on the type of iodination
sence of iodide and iodine monochloride. (enzymatic or nonenzymatic) and on the reaction conditions.
Enzymatic hydrolysates contained labeled hormone
and pyruvic acid; acid hydrolysates labeled thyronine EXPERIMENTALPROCEDURES
and acetic acid. (Treatment with acid converts hormone Preparation of [4C]Thyroglobulin-~-[U-4C]Tyrosine-labeled
to thyronine andpyruvic to acetic acid.) After borohy- hog thyroglobulin was prepared as described previously (8). It con-
dride treatment,labeled alanine was present instead of tained 4.4 m o l of newly synthesized iodine-free thyroglobulin/g of
pyruvic or acetic acid. The pyruvic acid/hormone, total thyroglobulin (2.9 pmol of [4C]thyroglobulin/nmol of thyro-
acetic acid/thyronine, alanine/hormone, and alanine/ globulin, corresponding to a molar ratio of 4 0 ) .
thyronine molar ratios always were 1,independently of Enzymatic Iodination of [4C]Thyroglobulin-Labeled thyroglob-
the method of iodination. ulin (3.3 mg, 5 nmol) was incubated at room temperature for 1% h
The coupling reaction consists of an oxidation step with potassium iodide (2.5 X M ) in the presence of thyroid
and nonoxidative coupling and decomposition steps. peroxidase (5 units) (19), glucose (6 mM), and glucose oxidase (Boeh-
The oxidation step may be either enzymatic or nonen- ringer, grade I, 5 units) in a total volume of 2.5 ml (50 mM sodium
phosphate, pH 7.4). The thyroglobulin was then precipitated with
zymatic. The decomposition step always leads to 1 de- ammonium sulfate (8).Iodination with lactoperoxidase (Sigma,L7129,
hydroalanine residue for each hormone residue synthe- 1 unit) was carried out similarly.
sized. (Dehydroalanine residues appear in the various Nonenzymatic Iodination ~f[~CJThyroglobulin-Iodine+ iodide,
hydrolysates as acetic acid, pyruvic acid, and alanine, iodide + chloramine-T, and iodine monochloride were used as iodi-
respectively.) Since proper alignment of 2 iodotyrosine nating agents.
residues is a prerequisite for coupling, a model is pro- A solution of iodine and potassium iodide in 2 ml of 50 mM sodium
posed according to which oxidation of hormonogenic phosphate, pH 7.4, or in 40 mM Tris-HC1, pH 8.5, was added slowly
iodotyrosine residues leads to a charge transfer com- (1h) with gentle stirring, a t room temperature or ina few experiments
plex which is the same zwitterion-biradical resonance a t 2 C, to a solution of 3.3 mg (5 nmol) of [4C]thyroglobulin in 1 ml
hybrid no matter whetherit resulted from a free radical of the same buffer. After another hour, the thyroglobulin was precip-
itated with ammonium sulfate (8). Concentrations of iodine, potas-
(enzymatic) or an ionic (nonenzymatic) oxidation. sium iodide, and thyroglobulin were 5 X M, 5 X M,and 1.67
X M, respectively.
A freshly prepared solution of chloramine-T in 1 ml of50 mM
The mechanism bywhich thyroid hormone residues are sodium phosphate, pH7.4, was added slowly (1h), with gentle stirring,
formed in thyroglobulin in the course of its iodination has at room temperature, to a solution containing [4C]thyroglobulin(3.3
mg, 5 nmol) and potassium iodide in 1 ml of the same buffer. After
been a controversial issue for the last 40 years. Various hy- another hour, the thyroglobulin was precipitated with ammonium
potheses (1-6) have been advanced as to the fateof the lost sulfate (8). Concentrations of potassium iodide, chloramine-T, and
side chain (7) which is eliminated in the course of the thyroglobulin were 2.5 X M, 1 X IO- M, and 2.5 x 10 M,
coupling reaction (formation of hormone residues). respectively.
In previously published experiments (8), iodination of [U- A solution of freshly redistilled iodine monochloride (1 pmol) in 3
14
Cltyrosine-labeled thyroglobulin was carried out in the pres- ml of 50 mM sodium phosphate, pH 5.5, was added slowly (1 h) at
2 cto 1 ml of a solution of [4C]thyroglobulin (6.6 mg, 10 nmol) in
ence of thyroid peroxidase. Labeled hormones (thyroxine and the same buffer. After another hour, the thyroglobulin was precipi-
3,5,3-triiodothyronine)and pyruvic acid were found in the tated with ammonium sulfate (8). Concentrations of iodine mono-
enzymatic hydrolysate of the iodinated [4C]thyroglobulin. chloride and of thyroglobulin were 2.5 X M and 2.5 x M,
The pyruvic acid/hormone molar ratio was found to be1, and respectively.
it was shown that the pyruvic acid is the only lost side chain Other experiments were carried out similarly at pH 7.4 and 8.1. In
these cases, the solution of iodine monochloride was brought from
The costs of publication of this article were defrayed in part by pH 5.5 to the desired pH by the addition of12 M NaOH and the
the payment of page charges. This article must therefore be hereby thyroglobulin in sodium phosphate solution also was a t the desired
marked advertisement in accordance with 18 U.S.C. Section 1734 pH.
solely to indicate this fact. Treatment of Iodinated [4CJThyroglobulin with Horseradish

9167
9 168 Mechanism of Thyroid Hormone Synthesis
Thyroglobulin
in
Peroxidase-The ammonium sulfate precipitate obtained in the io- Experimental Procedures, to the incorporation of roughly
dination of [4C]thyroglobulin with iodine monochloride at pH 5.5 the same amount of iodine in thyroglobulin (76-80 and 73-85
was dialyzed extensively at 2 C against 50 mM Tris-HC1, pH 8.5. atoms/molecule, respectively). The number of hormone resi-
Horseradish peroxidase (Boehringer, grade I, 0.5 unit), glucose (6
mM), and glucose oxidase (Boehringer, grade I, 5 units) were added to dues also was about the same no matter which method of
one-half of the retentate. After 1%h of incubation a t room tempera- iodination had been used (Table I).
ture, the thyroglobulin was precipitated with ammonium sulfate (8). The elution profiles obtained when the enzymatic hydroly-
Analytical Procedures-Treatment of [Clthyroglobulin with so- sates of various enzymatically or nonenzymatically iodinated
dium borohydride, enzymatic and nonenzymatic (HCl) hydrolysis of thyroglobulins wereanalyzed by cation exchangecolumn
[4C]thyroglobulin,and chromatographic fractionation of the various chromatography are shown in Fig. 1. The major peak repre-
hydrolysates on the cation exchanger AG50W-X4 were carried out as
previously described (8, 9). sents tyrosine since only a small fraction of the tyrosine
residues in thyroglobulin is being iodinated. The profile also
RESULTS shows major peaks of monoiodotyrosine, diiodotyrosine, and
hormone (thyroxine + triiodothyronine) in addition to several
Fate of the Lost Side Chain after Enzymatic and Non- known or unknown peaks. Among the known peaks is one
enzymatic Iodination of [4C]Thyroglobulin--Enzymatic io- representing pyruvicacidwhichwas identified by various
dination of newly synthesized, iodine-free [4C]thyroglobulin
previously described methods (8). Neither labeled serine nor
with either thyroid peroxidase or lactoperoxidase at pH 7.4
labeled hydroxypyruvic acid could, be detected (cf Ref. 8).
and nonenzymatic iodination with either iodine + potassium
Serine (2,5) and hydroxypyruvic acid (6) have been mentioned
iodide or with potassium iodide + chloramine-? at the same
as possible lost side chain products. The size of the pyruvic
pH led, under the experimental conditions described under
acid peak bears a direct relationship to that of the hormone
TABLE I peak (molar ratio = 1:l) no matter whether the thyroglobulin
Enzymatic and nonenzymatic iodination of [C]thyroglobulin
had been iodinated enzymatically or nonenzymatically. This
is also true for thyroglobulin which had been iodinated with
Newly synthesized iodine-free [4C]thyroglobulin prepared by in-
cubation of hog thyroid slices with [4C]tyrosine wasiodinated a t pH thyroid peroxidase (8).
7.4 and then hydrolyzed enzymatically. The hydrolysate was chro- It had beenshownpreviously (9) that the pyruvicacid
matographed on a cation exchange column (AG50W-X4) (Experi- found after iodination in the presence of thyroid peroxidase is
mental Procedures). The radioactivity of the eluted fractions con- derived from dehydroalanine residues formed in the course of
taining monoiodotyrosine, diiodotyrosine, and thyroid hormone (tri- the iodination. In order to determine whether or not the
iodothyronine + thyroxine), respectively, was counted and thenum- labeled pyruvic acid shown in Fig. 1 is always derived from
ber of iodoamino acid residues calculated on the basis of the specific
activity of the [U-4C]tyrosine (413 mCi/mmol) used in the prepara- dehydroalanine residues no matter whether enzymatic or non-
tion of r4Clthvroalobulin. enzymatic iodination has been used, we have now treated
Iodine in- [4C]thyroglobulin whichhad been iodinated with a variety of
corpo- iodinating agents with sodium borohydride. This was followed
rated in
Thyrox- newly by acid hydrolysis. The presence of dehydroalanine residues
Monoio- Diiodo- ine + tri- synthe- in thyroglobulin was revealed by the appearance of labeled
Type of iodination d:ig- tyrosine iodothy- sized io-
dotvro-
ronlne alanine in the hydrolysates. Labeled acetic acid was found
sines and instead of labeled alanine when treatment with borohydride
iodqthy-
ronlnes was omitted. (Acid hydrolysis converts pyruvic to acetic acid
residues/molecule thyroglobu- atoms/ as well as hormone to thyronine.)
lin molecule One example of borohydride treatment of [4C]thyroglobu-
Thyroid peroxidase 23 21 2.8 76 lin, which had been iodinated nonenzymatically (iodine +
Lactoperoxidase 41 8014 2.7 iodide), is presented in Fig. 2. The profiles obtained with
Iodine 22 21 2.3 73 noniodinated thyroglobulin (control) do not show a peak of
Chloramine-T 52 11 2.8 85 labeled alanine after treatment with borohydride nor a peak

m
I I
TYR
I
I
MIT DIT
I I
I
11

10

9
FIG. 1. Cation exchange chroma-
8 tography of hydrolysates of iodi-
nated @-Cltyrosine-labeled thy-
7 roglobulin. [C]Thyroglobulin (Table
PH I) was iodinated enzymatically (lactoper-
6 oxidase, -) or nonenzymatically (io-
dine + potassium iodide, - - - ) or chlor-
5 amine-? + potassium iodide - - -) at pH
7.4. The iodinated thyroglobulins were
4 hydrolyzed enzymatically and the hy-
drolysates chromatographed on a cation
3 exchange column (AG50W-X4) (Exper-
imental Procedures); pH gradient,
2
. . . ._

FRACTION NUMBER
 Mechanism of Thyroid Hormone Synthesis in Thyroglobulin 9169
I I I TYR I I 1 --
20-
FIG. 2. Cation exchange chroma-
tography of hydrolysates of iodi-
nated and noniodinated [Clthyro-
globulin before and after treatment
- with sodium borohydride. [14C]Thy-
- 15 -
0
roglobulin (Table I) wasiodinated (io-
iodide) dine + potassium and the iodi-
2 noniodinated
nated as well as the (con-
X hydrolyzed were trol) thyroglobulins
with hydrochloric acid before and after
L treatment with sodiumborohydride. The
2 10- hydrolysates
were
then
chromato-
u
f column exchange graphed on a cation
(Experimental Procedures). Iodinated
thyroglobulin before (-----) and
after
(-) borohydridetreatment; noniodi-
natedth-yroglobulin before (. - 1 and
after (- - -) borohydride treatment.

25 50 75 100 125 150

FRACTION NUMBER

of labeled acetic acid before such treatment. Hence, dehy- TABLEI1

droalanine residues are not present in newly synthesized io- Iodination of [4C]thyroglobulin with iodine monochloride
dine-free [4C]thyroglobulin. In contrast, labeled alanine is [4C]Thyroglobulin (TableI) was iodinated with iodine monochlo-
present in the hydrolysate of the iodinated [4C]thyroglobulin ride at pH 5.5,7.4, and 8.1,respectively. Theso treated thyroglobulins
which has been treated with borohydride and labeled acetic were then hydrolyzed and chromatographed as described in Table I.
acid is present when such treatment is omitted. This clearly [4C]thyroglobulin, iodinated with iodine monochloride at pH 5.5, was
also retreated at pH 8.5 with horseradish peroxidase (Experimental
shows that dehydroalanine residues have been formed in the Procedures).
course of the nonenzymatic iodination, together with hormone Iodine
residues. incomo-
Dependence of Hormone Synthesis on pH during Iodina- Thyox- rated in
newly
tion of [I4C]Thyrog2obulin with Iodine Monochloride- Type of iodination
Mono-
iodoty-
Diiodo.
tyrosine
ine +
triiodo-
synthe-
sized io-
When newly synthesized iodine-free [U-4C]tyrosine-labeled roslne dotwo-
dotyro-
thyroglobulin is iodinated with iodine monochloride at pH Ine sine: and
sines
5.5, 7.4, or 8.1, under otherwise identical conditions, an in- iodothy-
ronines
crease in pH resulted in an increased formation of monoio-
residues/rnoleeule thyro- atoms/
dotyrosine residues and in a decreased formation of diiodoty- globulin molecule
rosine residues (Table 11). This result is unexpected in view of Iodine monochloride, pH 5.5 19 31 0 81
the phenolic dissociation constants of tyrosine and of monoio- Iodine monochloride, pH 7.4 26 2069 0.73
dotyrosine. However, we have no data on the pH effects on Iodine monochloride, pH8.1 44 14 1.5 78
thyroglobulin conformation or on the concentration of the Iodine monochloride, pH 5.5, 18 31 1.5 86
iodinating species nor on the relative preference of iodine and then incubated with
monochloride for undissociated or dissociated phenols. There- horseradish peroxidase at
pH 8.5 (no iodide)
fore, the basis of the observed pH effect remains to be ex-
plained.
The total amount of iodine incorporated in thyroglobulin
remained roughly the same between pH 5.5 and 8.6 (about system (glucose-glucoseoxidase), but in the absence of iodide,
70-80 atoms/molecule). However, no formation of hormone at pH 8.5 (Table 11). Tn contrast to thyroid peroxidase or
residues took place at the acid pH, whilesome hormone lactoperoxidase, horseradish peroxidase has very different pH
residues were formed at the neutral pH andtwice as many at requirements for iodination and for coupling. No iodination
the alkaline pH (Table 11).This proves that the synthesis of takes place with horseradish peroxidase above pH 7, while the
hormone residues in thyroglobulin occurs in 2 distinct steps, pH optimum for coupling lies between pH 8.0 and 9.0 (14).
iodination and coupling, and that coupling is favored by an Furthermore, the presence of iodide is not required for cou-
alkaline pH. pling catalyzed by horseradish peroxidase (14), while coupling
The lack of coupling at an acid pH may be due to the catalyzed by thyroid peroxidase (19) or lactoperoxidase (20)
absence of hormonogenic iodotyrosine residues in thyroglob- does require the presence of iodide. Incubation at pH 8.5 with
ulin which had been iodinated with iodine monochloride at a horseradish peroxidase of [4C]thyroglobulin previously iodi-
low pH. It may also be due to the inability of hormonogenic nated with iodine monochloride at pH 5.5 resulted in the
iodotyrosine residues to undergo coupling at thatpH. In order formation of labeled hormone residues. The hormone yield
to check this point, [4C]thyroglobulinwas fiist iodinated with was the same as that obtained by iodination with iodine
iodine monochloride at pH 5.5 and then treated, after com- monochloride at pH 8.5. Consequently, hormonogenic iodo-
plete removal of iodine monochloride, with horseradish per- tyrosine residues were present after iodination of [14C]thyro-
oxidase, in the presence of a hydrogen peroxide-generating globulin with iodine monochloride at pH 5.5 and the lack of
 Mechanism of Thyroid Hormone Synthesis in Thyroglobulin
I I I I I
TYR MIT DIT

FIG. 3. Cation exchange chroma-

tography of hydrolysates of [C]
thyroglobulin treated with iodine
monochloride. [%]Thyroglobulin
(Table I) was treated with iodine mono-
chloride at pH 5.5 (-----), 7.4 (- - -), and
8.5 (-), respectively. The iodinated
thyroglobulins were then hydrolyzed en-
zymatically and the hydrolysates chro-
matographed on a cation exchange col-
PYRUVIC ACID
umn (Experimental Procedures).

25 50 75 100 125
FRACTION NUMBER

I I 0 hormone synthesis is clearly due to a lack of coupling at the

4 A acid pH.
The elution profiles of enzymatic hydrolysates of [%]thy-
roglobulin which had been treated with iodine monochloride
are shown in Fig. 3. It should be noted that the size of the
pyruvic acid peak is proportional to that of the thyronine
peak.
Stoichiometry between Hormone and Lost Side Chain
Product under Various Conditions of Iodination of [C]
Thyroglobulin-Fig. 4A gives the pyruvic acid/hormone mo-
lar ratio after enzymatic hydrolysis of [%]thyroglobulin and
1 2 3 4 Fig. 4, B and C, the alanine/thyronine and acetic acid/thyro-
HORMONE imol/mol Thyroglobulinl nine molar ratios, respectively, after acid hydrolysis.
Without treatment of thyroglobulin with borohydride, no
labeled alanine was found in the hydrolysates. Labeled pyru-
vie acid was found instead in all enzymatic hydrolysates and
acetic acid in all acid hydrolysates of iodinated [%]thyro-
globulin no matter which method of iodination had been used.
The acid hydrolysate of iodine-free [%]thyroglobulin (con-
trol) did not contain labeled acetic acid, indicating the absence
of dehydroalanine residues in iodine-free thyroglobulin. The
size of the pyruvic or acetic acid peaks always was proportional
to that of the hormone (or thyronine) peaks. The pyruvic
-1
acid/hormone (Fig. 4A) or acetic acid/thyronine (Fig. 4C)
1 2 3 4 molar ratios always were 1. Here again the method of iodina-
THYRONINE (mol/mol Thyroglobulinl tion is immaterial.
When iodinated [%]thyroglobulin was treated with boro-
4 c hydride before its hydrolysis, no labeled acetic acid was pres-
ent in the acid hydrolysates, but labeled alanine was found
3
0
(Table I) was iodinated enzymatically or nonenzymaticaby. It was
then hydrolyzed, after treatment with sodium borohydride or without
2
such treatment, either enzymatically or with hydrochloric acid. The
hydrolysates were analyzed by column chromatography (AG50W-
1 l7-l X4). The amounts of [%]pyruvic acid and of %-hormone were
determined after enzymatic hydrolysis (A) and those of [%]acetic
_--- +--io-QS--n-- acid. [Wlalanine, and [Clthyronine after acid hydrolysis (B and 0.
Treated with borohydride (---); no treatment with borohydride
3 4 (-). Type of iodination: thyroid peroxidase, pH 7.4 (m); lactoper-
THYROPiNE knokd Thymgbbulin)
oxidase, pH 7.4 (0); iodine + iodide, pH 7.4, 2 C (A); same, pH 7.4,
FIG. 4. Stoichiometry between hormone and lost side 20 C (0); same, pH 8.5, 20 C (~9; iodide + chloramine-T, pH 7.4,
chain product under various conditions of enzymatic or non- 20 C (0); iodine monochloride, pH 5.5, 2 C (0); same, pH 7.4 (0);
enzymatic iodination of [Clthyroglobulin. [4C]ThyroglobuIin same, pH 8.1 (+); control (noniodinated [4C]thyroglobulin) (0).
 Mechanism of Thyroid Hormone Synthesis in Thyroglobulin
instead. The alanine/thyronine molar ratio again was 1 (Fig.
4B).
The data clearly show (cf Ref. 9) that labeled pyruvic as
well as labeled acetic acid and labeled alanine are derived
from a common precursor which is dehydroalanine residues
and that1dehydroalanine residue is formed for each hormone
residue synthesized during iodination no
matter which method
of iodination (enzymatic or nonenzymatic) has been used.
DISCUSSION
When iodine + iodide or iodide + chloramine-T or iodine
monochloride are used as iodinating agents in aqueous media,
a number of ionic species are formed. These are interrelated
through a variety of reversible reactions, some of which are
shown in Scheme 1.
In addition to these species, hydrates or other complexed
iodine-containing species are present. The constants for the
various equilibria and, hence, the preponderance of one or the
other species depends on many factors such as pH, presence
or absence of other interacting ions, and presence or absence
of nucleophiles or electrophiles. For instance, at an acid pH,
iodine monochloride is likely to be principally in equilibrium
with I' (or hydrated 1') and C1- (Equation 6). I' is a strong
electrophile and, therefore, can iodinate nondissociated tyro-
sine residues, which explains why iodination with iodine
monochloride can proceed at anacid pH. In contrast,iodina-
tion with iodine + iodide does not occur at an acid pH,
possibly because negligible amounts of I' are present (Equa-
tions l and 2) under theseconditions. As previously shown by
Mayberry et al. (21 and Footnote l),the yield in the iodination
of tyrosine (or N-acetyltyrosine) with iodine + iodide depends
on the phenoxide anion concentration at a given pH.
The mechanism of the enzymatic incorporation of iodine in
tyrosine residues seemsto involve the formation of the iodine
radical, Io. This mayreact with the phenoxy radical of a
-
tyrosine residue in thyroglobulin or be further oxidized to I+
which then can react with the phenoxy anion of a tyrosine
residue (22, 23) (Equation 10).
-e- - e-
(101 l- l* + l+
Different mechanisms may also be operative in nonenzy-
matic and in enzymatic coupling. Hence, it could not be
assumed a priori that dehydroalanine residues are formed
during nonenzymatic iodination of thyroglobulin as well as
during enzymatic iodination.
In all nonenzymatic iodinations at a neutral pH, as well as
in iodinations with iodine monochloride at a neutral or alka-
line pH and in enzymatic iodinations in the presence of either
thyroid peroxidase (9) or of lactoperoxidase, dehydroalanine
residues were formed. Furthermore,the number of these
residues was always the same as that of hormone residues
' And preceding papers of the same series.
9171
formed in the same iodination reaction. This suggests a simi-
larity between the coupling mechanisms in enzymatic and
nonenzymatic iodinations.
It has been suggested by Johnson and Tewkesbury (1)and
by Harington (2) that in the oxygen-promoted conversion of
diiodotyrosine to thyroxine, dehydroalanine may be released
during the decomposition of a hypothetical quinol ether in-
termediate. This quinol either appears tobe extremely unsta-
ble in the free state (cf Ref. 24). If it were stabilized within
the framework of thyroglobulin, treatment with sodium bor-
ohydride followed by hydrolysis could not lead to the forma-
tion of alanine and thyroxine. Furthermore, hormoneresidues
in thyroglobulin can be determined spectrophotometrically
(15, 25). In thecase of the quinol ether, which is not a phenol,
the determination of aphenol-phenolate absorption shift,
which forms the basis of the spectrophotometric determina-
tion, is not possible. It can, therefore, be concluded that a
quinol ether intermediate, if formed, has a short lifetime.
The coupling reaction consists of a sequence of 3 steps:
oxidation of iodotyrosine or of iodotyrosine residues to an
activated form, actual coupling (formation of a covalent bond),
and fission of the coupling product leading to theformation of
hormone or hormone residues. Two or all 3 steps may take
place in rapidsuccession by a concertedmechanism. Thus, in
the hypothetical scheme of Johnson and Tewkesbury ( l ) ,the
1st step is the formation of phenoxy radicals, the second step
that of the quinol ether, and the third step its decomposition.
In enzymatic coupling, the H20e-activated enzyme is the
oxidative agent in the first step. Since coupling takes place
only in the presence of the enzyme and since peroxidases
promote 1-electron transfer reactions(26), it must be assumed
that 1-electron transfer reactions are involved in enzymatic
coupling. This does not, however, exclude the removal of 2
electrons from the same substratein rapid succession2which
appears more likely.
For the nonenzymatic coupling of iodotyrosine or its ana-
logs, the requirement for oxidizing agents is well known. No
thyroxine is formed when diiodotyrosine is incubated anaer-
obically (28,29), while aerobic incubation leads to thesynthe-
sis of thyroxine (30). In the nonenzymatic formation of hor-
mone residues upon treatment of thyroglobulin with iodine
+ iodide, the oxidizing agent is iodine in one form or another
(Scheme 1).Nishinaga and Cahnmann3 have shown that the
presence of oxygen is not required for this type of coupling.
Since the only conceivable source of free radicals in the
aerobic incubation is oxygen (which, in its triplet ground state,
has unpaired electrons in its molecular orbital), it was con-
cluded that iodine-promoted coupling is an ionic process and
does not involve free radicals.
Iodine monochloride-promoted coupling also appears tobe
an ionic process; here the oxidizing agent should be I' (or
hydrated 1') (Equation 6), which may react with a dissociated
or undissociated diiodotyrosine residue to produce an iodoty-
rosine residue cation. This cation must react with an iodoty-
rosine residue anion in order to form a hormone residue. A
neutral or alkaline pH is required for coupling (Table 11).At
this pH range, most iodotyrosine residues should be in the
anionic form.
When coupling is promoted by iodine + iodide, the oxidizing
agent must be either 11with delocalized o-electrons (I'+-I'-)
or also 1' (or hydrated I+) since IO- (Equations 3 and 4 )
cannot accept electrons from a phenolate anion. (Delocaliza-
An apparent 2-electron transfer reaction catalyzed by horseradish
peroxidase has been described by Claiborne and Fridovich (27).
Nishinaga, A., and Cahnmann, H. J., unpublished data presented
at the Annual Meeting of the European Thyroid Association, Jeru-
salem, 1972.
9172 Mechanism of Thyroid Hormone Synthesis in Thyroglobulin
tion occurs when 1 2 approaches the phenolate anion of an
iodotyrosine residue.)
A free radical mechanism has been posulated (1, 2) for the
oxygen-promoted conversion of diiodotyrosine to thyroxine.
However, also in this case, an alternativeionic mechanism (3)
has been proposed.
Both a free radical mechanism and an ionic mechanism
may explain the enzymatic coupling. The radical mechanism
implies that 1 electron is removed from each of the 2 iodoty-
rosine residues which undergo coupling. In the ionic mecha-
nism, 2 electrons are removed from 1 of the 2 interacting
residues, with formation of an iodotyrosine residue cation.
In approaching the question which mechanism is likely to
prevail in enzymatic coupling, the spatial requirements for
coupling must be taken intoconsideration. Thyroglobulin, like
other polypeptides, should be present in solution mainly or at
least partly in a preferred conformation (31) which is rein-
forced by numerous disulfide bridges.In such a conformation,
which should be different for different degrees of iodir,ation
(cf. Ref. 32), certain iodotyrosine residues (hormonogenicres-
idues) must not only be closeto each other in space, but also
face each other in such a manner (head-to-tail or nearly so)
that, upon oxidation, a charge transfer complex between the
2 coupling partners can be formed. These spatial requirements
are suggested by previously reported model reactions (33).
In Scheme 2 , a model is presented which is based on our
present and previous experimental findings. According to this
model, the withdrawal of 2 electrons from a pair of hormon-
d+ d- I
I+or 1-1 l 2050-2055
I -0
J- f
I T
HC-C-
L C A 4
I 25. Covelli, I., Van Zyl, A,, and Edelhoch, H. (1971) Anal. Biochem.
f 'W0-
SCHEME
2
ogenic iodotyrosine residues, which in the case of enzymatic
oxidation should occurin 2 successive 1-electron transfer
steps, will lead to the formation of a charge transfer complex.
This complex is a zwitterion-biradical resonance hybrid of
which only1 of many limiting resonance forms4is shown. The
quinol ether intermediate is the same which had already been
proposed by Johnson and Tewkesbury (1) and by Harington
(2).
The postulated charge transfer intermediate is the same
zwitterion-biradical resonance hybrid no matter whether the
oxidation step leading to itis an ionic ora free radical process.
Therefore, it is not surprising that, as found in the present
investigation, the same final products, hormone residues and
dehydroalanine residues, are found in all enzymatic as well as
nonenzymatic coupling reactions.
Acknowledgments-We gratefully acknowledgethe valuable tech-
nical assistanceof Pierrette Menuelle. H. J. C. thanks the Fondation
pour la Recherche Medicalefor financial support.
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