Iodothyronine 5' deiodinase in rat kidney microsomes Kinetic behavior at low substrate concentrations

Iodothyronine 5'-deiodinase in rat kidney

microsomes. Kinetic behavior at low substrate

concentrations.

A Goswami, I N Rosenberg

J Clin Invest.

1984;74(6):2097-2106. https://doi.org/10.1172/JCI111634.

The thiol-activated enzymatic outer-ring monodeiodination of iodothyronines by rat kidney

microsomes at low (nanomolar) substrate concentrations shows an apparently sequential

reaction mechanism and is further characterized by insensitivity to inhibition by dicoumarol,

a moderate sensitivity to inhibition by propylthiouracil (Ki = 100 microM) and iopanoic acid

(Ki = 0.9 mM), responsiveness to 5 mM glutathione (GSH), and a thermal activation profile

that is concave downward with a Td of approximately 20 degrees C. In contrast, the activity

at high (micromolar) substrate concentrations shows a ping-pong reaction mechanism, is

inhibited by micromolar concentrations of propylthiouracil, iopanoic acid and dicoumarol, is

unresponsive to 5 mM GSH, and shows a concave upward thermal activation profile.

Analysis of the microsomal deiodinase reaction over a wide range of 3,3',5'-triiodothyronine

(rT3) concentrations (0.1 nM to 10 microM) suggested the presence of two enzymatic

activities, with apparent Michaelis constants (Km) of 0.5 microM and 2.5 nM.

Lineweaver-Burk plots of reaction velocities at nanomolar substrate concentrations in presence of 100

microM propylthiouracil also revealed an operationally distinct enzymatic activity with Km's

of 2.5 and 0.63 nM and maximum velocities (Vmax's) of 16 and 0.58 pmol/mg protein per h

for rT3 and thyroxine (T4), respectively. These findings are consistent with the presence of a

low Km iodothyronine 5'-deiodinase in rat kidney microsomes distinct from the well

characterized high Km […]

Research Article

Find the latest version:

lodothyronine

5'-Deiodinase

in

Rat

Kidney Microsomes

Kinetic

Behavior at Low

Substrate

Concentrations

AjitGoswami and Isadore N. Rosenberg

DepartmentofMedicine, Framingham UnionHospital,

Massachusetts 01701; Department ofMedicine, Boston University Schoolof Medicine, Boston, Massachusetts 02118

Abstract.

The thiol-activated enzymatic

outer-ring monodeiodination of

iodothyronines by

rat

kidney

microsomes

at

low

(nanomolar)

substrate

concentrations

shows

an

apparently sequential

reaction

mechanism and

is further characterized by

insensitivity

to

inhibition

by

dicoumarol,

a

moderate

sensitivity

to inhibition

by

propylthiouracil

(Ki

=

100

MAM)

and

iopanoic

acid

(K1

=

0.9

mM),

responsiveness

to

5

mM

glutathione (GSH),

and a thermal

activation profile

that is

concave

down-ward

with

a Td

of

-20'C.

In contrast,

the

activity

at

high (micromolar)

substrate

concentrations

shows a

ping-pong

reaction

mechanism,

is inhibited

by

micro-molar concentrations of

propylthiouracil, iopanoic

acid

and

dicoumarol, is unresponsive

to

5 mM

GSH,

and

shows

a

concave

upward

thermal activation

profile.

Analysis of

the

microsomal

deiodinase reaction

over a

wide

range

of

3,3',5'-triiodothyronine (rT3)

concentrations

(0.1

nM

to

10

uM)

suggested

the presence of

two

enzymatic

activities, with

apparent

Michaelis

constants

(Kin)

of

0.5 ,M and 2.5 nM.

Lineweaver-Burk plots of

reaction velocities

at

nanomolar substrate

concentrations

in presence

of 100

,M

propylthiouracil

also revealed an

operationally

distinct

enzymatic activity

with Km's of

2.5 and 0.63 nM and

maximum velocities (Vmax'S) of

16 and

0.58 pmol/mg

protein

per h

for

rT3

and

thyroxine

(T4),

respectively.

These

findings

are

consistent

with the

presence

of

a

low

Km

iodothyronine 5'-deiodinase

in rat

kidney microsomes distinct from

the well

characterized

high

Km

enzyme and

suggest that at

circulating levels of

free

T4 the postulated low

_K.

enzyme could be

physio-logically important.

Receivedfor publication 5 March 1984 and in revised form 26 July 1984.

Introduction

Whereas the

thyroid

gland is the sole source of

circulating

thyroxine

(T4),'

3,5,3'-triiodothyronine (T3),

the

biologically

active form of the

thyroid hormone,

is

largely

generated by

5'-monodeiodination

of T4

extrathyroidally.

Theenzyme system responsible for this conversion has been identified in the microsomal fractionsof the rat liverand

kidney

and

partially

purified

(1, 2). The microsomal enzyme has an apparent Michaelis constant

(Ki)

of -5 MM for T4, is activated

by

thiols, especially

dithiols,

in a two-step transfer

(ping-pong)

mechanism, is inhibited by

propylthiouracil

and can use

reverseT3

(3,3',5'-triiodothyronine,

rT3)as analternatesubstrate

(Km

- 0.5

AM)

(3).

AlowKm

(10-9

Mfor T4) iodothyronine 5'-deiodinase has recently been described in rat brain (4), pituitary (5), and brownadiposetissue (6). Theenzyme(designatedtypeII) also usesrT3 as an alternate substrate with high affinity

(K.

- 3 X

10-9

M), butisdifferent from thehigh

_K.

(type I)hepatic and renal enzyme in being insensitive to propylthiouracil (PTU)andinits thiol activation mechanism, which is believed to be sequential rather than ping-pong (5). This type II

enzymatic

pathwaymay be

particularly

importantinregulation of intracellular, as opposed to circulating triiodothyronine levels, but may also play a role in the generation of that fraction of extrathyroidally produced T3 that is not blocked byPTUin athyreoticT4-replaced rats (7). Based on inhibition analysis with PTU, Silva and co-workers (7) also concluded thatthe type IIpathway,while ofmajor importance in brain and pituitary, wasof little, ifany, quantitative significance in the ratliver.

Inthe present study, we have

examined

thekinetic behavior ofrat kidney microsomal iodothyronine

5'-deiodinase

at low (nanomolar) concentrations of the substrates, T4 and rT3. The experiments revealed some properties of the enzyme at low substrate concentrations that differed significantly from those at high substrate concentrations and, indeed, resembled in

1. Abbreviations used in this paper: DHL, reduced lipoamide; DTT, dithiothreitol; GSH, glutathione; PTU, propylthiouracil; rT3, 3,3',5'-triiodothyronine; T3,3,5,3'-triiodothyronine; T4, thyroxine.

2097 RatKidney Iodothyronine 5'.Deiodinase

J.Clin. Invest.

© The American Society for Clinical Investigation, Inc. 0021-9738/84/12/2097/10 $1.00

some,butnotall, respectsthe characteristics described forthe

type II enzyme in the cerebral cortex, pituitary, and brown adipose tissue. We report here some observations on kidney microsomal 5'-deiodination of T4 and rT3 at low substrate

concentrations and raise the possibility, on the basis of kinetic and other data, that rat kidney microsomes may contain both alowand a high Km iodothyronine 5'-deiodinase.

Methods

Reagents. L-[3'- or

5'-'25-I]

rT3 and T4 (750-1,250

_gCi/Mg)

were obtained from New England Nuclear (Boston, MA) and were further purified just before use by high voltage paper electrophoresis (3). Dihydrolipoamide (DHL) was prepared by borohydride reduction of theoxidized form (obtained from Sigma Chemical Co., St. Louis, MO) by the method of Reed et al. (8) as modified by Silver (9). lopanoic acid (Telepaque) was a product of Winthrop Laboratories Co., New York; a stock solution was made by alkaline extraction of the tablets andneutralized before use.

Assay ofmicrosomaliodothyronine S'-deiodinase. Rat renal micro-somes were prepared by methods as described earlier (10). The incubation medium contained, in a total volume of 200

_;l:

microsomal protein, 50 Mg; K-phosphate buffer (pH 7.0), 20 Mmol; EDTA, 200 nmol; and varying amounts of dithiothreitol (DTT), rT3, or T4 (containing -50,000 cpm of '25I-labeled rT3 or T4), as indicated in legends. The incubation times were 3 min for rT3 (except in the experiment of Fig. 1) and 60 min for T4. The incubations were terminated by the addition of 0.1 ml of 2% bovine serum albumin (BSA), followed immediately by 0.5 ml of 10% ice-cold trichloroacetic acid. '25I- liberated was measured after separation by ion-exchange chromatography on Bio-Rad Ag5OWX8(H+) resin (BioRad Labora-tories,Richmond,CA)asdescribed previously (10). The reaction rates wereexpressed as picomolesofrT3 degraded per milligram protein per hour. The products were frequently checked by descending paper chromatography (solvent system-hexane/t-amyl alcohol/ammonia (2 N) 1:10:11).

Results

Studies

of

microsomal 5'-deiodination over a wide range

of

substrate concentrations. Fig. 1 showsrT3deiodinaseactivities ofkidney microsomes, at5mMDTT,from agroupof normal

rats and from rats given intraperitoneal PTU (2

mg/l00

g body wt) 15 min before killing. A plot of enzyme reaction

velocities vs.-log

(S]

for normal microsomeswassigmoid (Fig. 1 A), withan inflection at M0.5

gM

andamaximumvelocity of -58 nmol/mg protein per h, thus reflecting the

well-documented characteristics of the kidney 5'-deiodinating

en-zyme. In a plot ofthe activities at substrate levels <10 nM

and

extending

down to 0.1 nMon a 25-fold expanded scale, however, adeviationwasnoted between theobserved reaction

velocities andthose

predicted

bythe Michaelisequation, using valuesforKmand

V.,.

of 0.5 ,uMand 58 nmolrT3 degraded/ mg protein perh,

respectively,

asobtained from experiments athigher substrateconcentrations for this microsomal prepa-rationjust before this

experiment.

Asshown in

Fig.

1 A, the

observed reaction velocities over the range of substrate

con-p[S]

Figure 1.Semilogarithmic

110 B plots of reaction velocities

100 vs. rT3concentrations

ob-100 < _{tainedwith renal}

micro-80 somesfromnormal and

> \ PTU-treated (2 mg/100 mg

60

60 bodywt, i.p., 15

min

be-40- fore killing)rats.The

incu-bation medium contained,

20- _{in a total volume of 200 Ml:} ° 7₇ 8 9 10_L_ microsomal

protein,

50

Mg;

[

9J

_K-phosphate

_buffer

_(pH

7.1), 20

Mmol;

EDTA, 200 nmol; DTT, I

gmol

andvaryingamountsofrT3(containing 50,000

cpmof'251-rT3),asindicated. The incubation wasfor I min andwas

terminated bytheaddition of 0.1- ml of 2%BSA,followed immedi-atelyby0.5 mlof 10% TCA. I-liberatedwasmeasuredbyion

exchange,

chromatographyonBio-RadAG50WX8resinasdescribed (10). (A)Activities in normal(-)and PTU-treated(o)microsomesas

afunctionoflog

l/[rT3].

The lower part ofAshows the activitiesat

nanomolar substrateconcentrationson a25-foldexpandedactivity

scale. Also shown(dashedline)isatheoreticalplotof theactivities,

atlow(nanomolar)substrateconcentrations, extrapolatedfrom the kinetic parameters observedathigh(micromolar)substrate

concen-trations. The inset shows doublereciprocal plotsof theactivity profileat0.75-20-nM substrate range.(B)Difference between

reac-tionratesobservedatlow

[S]

and those

projected

from the kinetic parametersathigh 1S],plottedas afunction of_log

l/[rT3].

The

reactionrateswerecalculatedaspicomoles rT3 degradedper milli-gramproteinper hour. The dataarefromone

experiment,

done in

duplicate,andarerepresentativeof three similar

experiments.

centrations from 0.25 to 10 nM (solid line)were consistently greaterthan the extrapolated values (dotted line) and showed a sigmoidal profile.2 The Lineweaver-Burk plots also showed a discontinuity at the 5-10-nM substrate range, as shown in the inset. A difference curve, constructed by plotting the difference between observed andpredictedvalues of enzymatic activity at the low substrate concentrations, also showed a sigmoidal profile (Fig. 1 B) suggesting the presence ofa low Km enzymatic activitywith aKm of -2.5 nM and a

_V.

of

-1 10 pmol rT3 degraded/mg proteinper h.

Thisanomalousbehavior of the enzymecannotbeexplained on the basis of increased fractional binding of substrate to

microsomal proteins at low substrate

concentrations,

as the reaction velocity wouldbeexpected to

decrease,

notincrease, because ofdiminished availability of substrate tothe enzyme under such conditions. Moreover, under the conditions used in these experiments, binding of

iodothyronine

substrate to

microsomal proteinswasfoundtobe

relatively slight

(5-10%) and valuesfor percentage

binding

werewithin the same range atboth micromolar and nanomolarsubstrateconcentrations.3 The activity profile of the microsomes from the PTU-treated rats showed the expected sharp decline in

activity

at

substrateconcentrations above 100 nM,

confirming

the findings of Leonard and Rosenberg (3), with relatively less inhibition (35-40%)atlower(0.5-2.5 nM) substrate concentrations. The plotofthePTU-inhibited activity wasdoublysigmoid(Fig. 1 A) and, while the activity at the

higher

range of substrate concentrations had a

_Km/

_V.

ratio similar to that obtained with normal microsomes at high substrate concentrations, at the lowsubstrateconcentration avalueforKm (-2.5 nM)was observedthatwassimilartothatderivedfromtheuninhibited profileatthisrange, butwithalower V,, .Whiletheinhibition pattern at high substrate concentrations is as expected from the well known

uncompetitive

characterofPTUinhibition (3, 11, 12)of therenal andhepatic enzyme, theinhibition profile at low substrate

concentrations,

characterized by lesser PTU-sensitivityand an unaltered

K.,

indicates eithersome unusual

2Reproducibility

of data. For the points in Fig. 1 _{A at which the}

substrate concentrations were <1

IMM,

the observed values for rT3

degradationranged between 11.7 and 34% of the total and the absolute differences between duplicate determinations were usually <1 and in

nocase >2.8; the deviations of the duplicates from the means averaged 4%. Forthe 1-, 2.5-, and 5-MM substrate concentrations, the mean percentagesof rT3 degraded were 8.1, 4.2, and 2.2%, and the duplicates differed by 1.0, 0.4, and 0.6, respectively.

3Thebindingstudies were performed by incubation of renal microsomes (50Mgprotein) with 0.5 nM and 5AM labeled rT3 or T4 at 41C for 15 min in 200 Ml of0.1 M potassium phosphate buffer (pH 7.0) followed by ultrafiltration through Millipore HAMK filters (Millipore Corp., Bedford, MA), with analysis of the radioactivity in both the filterand the filtrate. Nonspecific binding was determined after incu-bation in presence of a 100-foldexcess of nonlabeled rT3 or T4 and

wasbetween 2 and 3% of total radioactivity. Under these conditions

the specific binding for rT3 and T4 was 8 and 6 fmol at the low and

17 and 11 pmol at the high substrate concentrations, respectively.

kineticsofa PTU-modified enzyme atlow substrate

concen-trationsorthe presence ofa separate lowKm enzyme with a

different mechanism of inhibitionbyPTU.

ThevaluesforKmand

V,,,,

asdetermined from the linear portionsof theLineweaver-Burk plots at low substrate

concen-trations in presence of 100 AM PTU were 2.5 nM and 16 pmol/mg proteinper h for rT3 and 0.63 nM and 0.58 pmol/ mg protein per h forT4, respectively, at 2.5 mM DTT. The DTTconcentration of 2.5 mM was a compromise to achieve

a level of enzyme activity that was detectable by the assay procedure used, yet was not high enough to overcome the inhibitory effects of PTU, especially at high substrate concen-trations(10-12). Theproducts ofthe enzymatic reactionwere

identified by paper chromatography as 3,3'-diiodothyronine and I-, and T3 and I-, in equimolar proportions, from rT3 and T4, respectively. The reaction rates were proportional to the amounts of microsomal protein and the enzymaticactivity wascompletelyeliminated by heat treatment

(80'C

for 1 min) orby pronase digestion of the microsomes before incubation. Athigh substrate concentrations(range, 0.1 to 10 ,uM), in the absence of PTU, the kinetic parameters for T4 and rT3, as determined by Lineweaver-Burkplots,were:Km(rT3), 0.5 AM; Km(T4), 5

AM;

Vmax(rT3), 60 nmol; and

V.

(T4), 2.1 nmol/ mg protein per h, respectively, in reasonable agreement with published values for kidney microsomes (3); these activities werecompletely inhibited at 100

AM

PTU.

The doubly sigmoid substrate concentration curves, ob-served in both normal and PTU-treated microsomes (Fig. 1, AandB), indicate the presence inrenal microsomesof either (a) a single enzyme withnegativelycooperative ligandbinding, or (b) two enzymes with different Km values acting on the samesubstrate (13). This question was then addressed experi-mentallybyinhibition and activation analyses of the enzymatic activities at micromolar and nanomolar substrate

concentra-tions.

Inhibition analysis. PTU inhibited the renal microsomal enzyme, when assayed at 0.5 nM rT3, by 30% at 100

_AsM;

this was considerably greater than one would expect from the inhibition constant

_(Ki)

(1 MM) reported, and experimentally confirmed, for the uncompetitive inhibition ofthe high Km enzyme. As shown in Fig. 2, the inhibition was characterized bypartial inhibition kineticswith -90% inhibitionofthe total activity at 1 mM PTU, and very little further inhibition at higher concentrations of the inhibitor. The data suggest satu-ration ofa separateinhibitor-bindingsite.The residual activity in presence of excess of inhibitor was near the limits of experimental detectionandwas not furtheranalyzedkinetically. In contrast to PTU, iopanoate and dicoumarol inhibited -65%of theactivityat 10MM.4The residual activity, however,

was more resistant to these inhibitors, requiring -2.5 mM 4Thisdegree ofinhibition is close to what one would expect from 10 MMiopanoate (K, =9.2MM)if itwereinhibiting onlythat component of the enzymatic activity attributable to the high Km enzyme as determined by extrapolation(e.g.,as inFig. I A).

60-

(50-C

5,

40-0

30

20-

10-0*

0.2 0.4 0.6 0.8 1.01.5 2.5 3.0

[11mM

Figure 2. Effects ofvarious concentrations of PTU(.),iopanoate (o), anddicoumarol (a)onmicrosomal 5'-deiodinationof rT3. The

ab-scissareferstomillimolar concentrations of the inhibitor. The micro-somes wereincubatedfor3 min in absence or in presence of indi-catedconcentrationsofspecified inhibitors under conditions as de-scribed inFig. l. rT3 was 0.5 nM and DTT at 5 mM. The values are from one of three closely agreeing experiments.

iopanoateforcompleteinhibition, while dicoumarol was much less active (40% inhibition at 2.5 mM). The results indicate thepresenceofaseparateenzymewith different sensitivity to these inhibitors.

Lineweaver-Burkplots at nonsaturating PTU concentrations (Fig. 3 A) showed noncompetitive, in contradistinction to uncompetitive, kineticsover the rangeoflower rT3 concentra-tions (0.25-2.5 nM). A replot ofthe vertical axis intercepts against the inhibitor concentrationswaslinear (Fig. 3 A,inset) andrevealeda

Ki

of- _{I00MM, comparedwithaK, of 1}

_;M

reported for the uncompetitive inhibition by PTU at high substrate concentrations (14). The inhibition by PTU was competitive with DTT (Fig. 3 B),withconsiderable releaseof the

inhibition

at high DTT concentrations. For example, at 550 MM PTU,

although

the inhibitionwas -87% at2.5 mM DTT,itwasonly -56% at 20 mM DTT. Arelativeinsensitivity was also observed with

iopanoic

acid, a potent inhibitor of microsomal deiodination of

iodothyronines

at high substrate concentrations (15).The inhibitionby iopanoatewas compet-itive withrT3

(Fig.

4),asisthecasewiththe

high

Km enzyme, but the observed

Ki (0.9 mM)

was over 100-fold greater than thatreported for the

high

Km enzyme

(16).

Similar

inhibitory

patterns [K;

(PTU),

100

MM; Ki (iopanoate),

1.38

mM]

were

also obtained withthe 5'-deiodination of

T4,

when

assayed

at

nanomolar substrate concentrations, and the

similarity

ofthe

K,'s

suggestedthat theouter

_ring

deiodination of both substrates

N

.5 06

01

E

N,

01

a

*0

-0.06

-600

rT3(nM)-'

.C

80.4

E /0.5

0.3/

id 21-0 2

E 02

0.1 0.2 0.3 0.4.

DTTimMr'

Figure 3. Doublereciprocalplots of PTU inhibitionof

rT3-deiodi-naseactivitywith(A) rT3asa variable substratewithDTTat5mM,

and(B)DTT as avariable substrate withrT3at0.5nM.Incubation was for 3 min.ThePTU concentrationschosenwerenonsaturating

(compareFig. 2), and,inadditiontothe indicated

concentrations,

all

incubationswereperformedat abasal PTU level of 50 MM.An

intercept replot(A,-inset)waslineargivingaKiof 100MMfor the inhibition. The dataarefromasinglerepresentativeof three

closely

agreeingexperiments.

at these concentrations was mediated

by

the same enzyme. The renal enzyme,

assayed

at low substrate

concentrations,

thus differs from the type

II,

low Km

pituitary

and brain

lopanate

(mM) op1.0

c ._

0

E

0

0

0

l-0

t

0 2 4

K;=0.9mM rT3(nM)-'

6 8 1U

Figure4.Competitiveinhibition ofrT35'-deiodinaseby iopanoic acidatnanomolar substrateconcentrations. Incubationwasat50 uM

PTU and 5 mM DTT. Incubation timewas3min.AKi of 0.9 mM

wascalculated for the inhibition. The dataarefromoneof three

similarexperimentswhere thevaluesagreedwithin 10% of each

other.

enzymes in being moderately, ifincompletely, PTU-sensitive

andthismayaccountfor thefailure of Silva et al.(7)to detect alowKmenzymebyinhibitionanalysisalone.

Progress curves. Fig. 5 shows the progress curves of the

enzymatic activity at 0.5 nM rT3 in the presence of 50 and

150

gM

PTU. The profiles resembled that reported for the

highKmenzyme(3),composedofaninitialrapid phase during the first minute, followed by a slower phase of enzymatic activity. The activity at 150 MM PTU was -40% inhibited

from the levels obtained at 50 MM PTU. This inhibition, however, was observed in both the early and later phases of

theprogress curve,in contrast tothehighKmprogress curve,

where the rapid early phase, presumably representingthe first

catalytic cycle, has beenreportedtobe insensitivetoPTU(3). These findings indicate that the 5'-deiodination of rT3 at

nanomolarconcentrations is steady andsustained during the entire incubationperiodandthat the relativePTU-insensitivity

cannotbeattributedtotheenzymeactivity duringthe brief(1 min) period of PTU-insensitive catalytic activity.

Reaction mechanisms. Lineweaver-Burk plots of reaction velocities vs. rT3 concentrations in the nanomolar range at

fixed DTT concentrations and in presence of 50 MM PTU

generatedaseriesoflinesthatintersected atthe velocityaxis (Fig. 6 A), in sharp contrast to the parallel lines observed at

micromolar substrate concentrations (inset). A similarplot of

reaction velocitiesvs.DTTconcentrationsatfixedrT3 concen-trations yielded a series of lines intersecting in the second

Time(min)

FigureS. Progresscurvesof microsomalrT35'-monodeiodinasein 50

MM (o)and 150 MMPTU(o)at0.5 nMrT3.The microsomal preparations and theincubation conditionswere asdescribed inFig.

1.Thedataarefromoneoftwoexperimentsincloseagreement.

quadrant above the I/Saxis (Fig. 6 B) and a secondary plot (inset) of the slopes of these lines against l/rT3 yielded a

straight line passing through the origin (intercept zero). The

data suggestanorderedsequentialmechanismfor the deiodinase

reactionand wouldseemtoindicate the formation ofaternary

complex in which rT3 is the second substrate (17, 18), in contrast tothe well documentedping-pongmechanismathigh

substrate concentrations.

Thermal activationprofiles. The effectsoftemperature on

the

_V.

of the enzymatic reaction, when measured at low

(nanomolar)substrateconcentrationsinpresenceofPTU, also

differedsignificantlyfromthose observedathigher(micromolar)

substrateconcentrations in absence of PTU. As shown in Fig.

7, while the Arrhenius plots of both activities showed

discon-tinuities (Td)at '20'C, the display was concavedownwards whenmeasuredatalow substrate concentration (20nM)with

activationenergies of 5.98 and 11.8 kcal/mol above and below

the Td,respectively. This wasin sharpcontrast to theprofile

obtained with 5 ,uM rT3, which was concave upwards with

activation energies of 2.14 and 7.5 kcal/mol, respectively,

above and belowthe Td.

Activation energiesareregardedasintrinsic characteristics

of enzymes rather than substrates (19), and the different

thermal activation profilesobservedathigh and lowsubstrate

concentrations can be taken as indirect -evidence for two separate enzymes operating at these two substrate levels. Itshouldbenoted, however, thatthereaction velocitiesatthe

lowsubstrate levelweremeasuredinpresenceofPTU andthe

possibility that the activation profile seen at this substrate

concentration was that ofa PTU-modified form ofa single

2101 RatKidneyIodothyronine S'-Deiodinase

C

-W 0

E

IN 0 0 0

rT3,20nM

1.5

1.4

1.3

1.2

1.1 1.0

0.9

0.8

3.2 3.3 3.4 3.5 3.6

1OOO/T

3.2 3.3 3.4 3.5 3.6

1OO0/T

Figure 7. Temperature activation profiles ofmicrosomalrT3

5'-mon-odeiodinationatmicromolar and nanomolarsubstrate

concentra-tions. Microsomes (50 jigprotein)wereincubated with5jiM(A)or

20 nM (B) rT3 inpresenceof5 mM DTTattheindicated

tempera-turesfor3 min, and theratesof deiodinationweremeasured by

methodsdescribed in Fig. 1.In B the incubation mediumalso

contained 50 juM PTU. The reaction velocitieswerecalculatedas

picomoles rT3 degradedpermilligram proteinperhour. Thedataare

fromarepresentative of three closely agreeing experiments.

rT3 (nM)-'

E)TT (mM)-l

Figure6.Renalmicrosomal rT35'-deiodinaseactivitiesatnanomolar

substrate concentrationswith(A)DTT and(B) rT3asfixed

sub-strates.Incubationtimewas3 min.InA,activity patternsobserved

atmicromolarrT3concentrationshavebeenincluded for

compari-son.In B,aslope replot (inset)waslinearpassing throughtheorigin. All incubationswereperformedin50AMPTU.The data arefroma

representativeof threecloselyagreeing experiments.The values

agreedtowithin10%of eachother.

enzyme cannot be entirely excluded. The linearity of the

Arrheniusplotwithin thetemperaturerangesunder observation

would, however, suggest thattheoverallvelocityiscontrolled

byasingleratelimitingstepthroughoutthetemperaturerange (20) and that its thermal activation coefficient isunaffectedby interaction withPTU.

Relative

substrate preference athigh and low

_[S].

Table I shows the

5'-deiodinase

activities of rat kidney microsomes with0.5 nM rT3 and T4 in presence of DTT and DHL. In the presence of5 mM DTT and DHL, while the rT3 deiodinase

activities

were only slightly abovethe values calculated from thekinetic parameters derived from experimental values ob-served with high substrate concentrations, the

T4

deiodinase activitieswere about fivefold higher. The preferred substrate for the

deiodinating

reaction at low substrate concentrations thus appears tobe T4ratherthanrT3. This isalso borne out

TableI.

S'-Monodeiodination

ofT4andrT3(0.5 nM) by KidneyMicrosomes:

Effects of

Various Thiols

Y-Monodeodinaseactivity

(pmolsubstratedegraded/ngproteinperh)

Thiol T4 rT3

DTT 1.1±0.15 (0.21) 93.4±7.5(60)

DHL 1.4±0.18 (0.21) 104.8±9.7(60)

Eachfigurerepresentsmean±SE ofdata from sixexperimentsin whichrT3was substrate and four in whichT4wassubstrate,each done induplicate.Assay conditionswereasdescribedin thetext.

Thefiguresinparenthesesrepresenttheoreticalvalues calculated

fromthefollowing kineticconstants(seetext):Km(rT3),0.5

AM;

Km

(T4),

5AM; V (rT3),60; and

_V.,,,

(T4), 2.1

nmol/mg

proteinper h.

2102 A. GoswamiandI.N. Rosenberg.

5.0

4.8 x

* 4.6

E

>4.4

0

4.2

4.0

3.8

A B

by the lower Km for T4 compared with rT3 observed at low substrate concentrations (see above) and is a reversal ofthe relativesubstrate preference seen athigh substrate concentra-tions.

Thiol activation profiles. The thiol activation of enzymatic activity at nanomolar substrate concentrations was further investigatedover awiderange ofconcentrations ofglutathione (GSH), DTT, and DHL, with results as shown inFig. 8. The previously reported 10-fold greater potency of DHL than of DTT (14) instimulating deiodinating activityathighsubstrate concentrationswasalsofoundatnanomolarconcentrationsof substrate. In addition, while the enzyme, in agreement with published data (14), was almost totally unresponsive to 1-5 mM GSH at high substrate levels, it responded to these concentrations ofGSH when rT3 was 0.5 nM, at -20-25% of the level with DTT.Although thenatureof this differential GSH-responsiveness athigh andlow substrate concentrations is unclear, a possible explanation may be that rat kidney microsomes may contain an operationally distinct GSH-sen-sitive lowKm enzyme.

A summary of the kinetic parameters ofthe enzymatic activityathigh andlow substrate concentrations is presented inTableII.

Discussion

When assayed at low (nanomolar) substrate concentrations, rat kidney microsomal iodothyronine 5'-deiodinase displays somekinetic characteristicsthatdiffer significantlyfrom those observedathigh (micromolar) substrate concentrations. Some of these

differences,

notablytherelativePTUinsensitivityand asequential reaction mechanism observedat low(nanomolar) substrate concentrations asopposedtothehigh

PTU-sensitivity

and thetwo-steptransfer (ping-pong) mechanism, whichhave been observed athigh (micromolar) substratelevels, are rem-iniscent ofthe low and high

Km

enzymes described in the

iff rT3, 0.5 nM A rT3, 0.5uM

2 100 _DHL

E

E DiT

so

60

C

.2

40-S~ ~ ~ ~ ~~~~~S

20

0

5 4 3 2 17 6 5 4

p[-SHJ p[-SH]

cerebralcortex

(4), pituitary (5),

and brown fat

(6),

and would suggest the presence also of a distinct low

Km

enzyme in

kidney

microsomes. Thereare,

however,

several

considerations,

as discussed

below,

that must be

critically

evaluated before suchan

_{interpretation}

canbe made.

Inhibition kinetics.

(a)

PTU inhibition of the enzyme at micromolarsubstrate concentrations ischaracterized

by parallel

upward displacements

of double

reciprocal plots (3).

From the kinetic expression of such uncompetitive inhibitions (21,

22):

v =

_Vma,/[(Km/S)

+

₍₁

+

i/Ki)],

where i and

Ki

refer to the inhibitorconcentration and theinhibitorconstant,

respectively,

it is clearthat inhibition at any fixed concentration of PTU would become progressively lowerassubstrateconcentrations

arelowered becausethe

i/Ki

termin thedenominator becomes small in relationto

KnrS.

For

example,

if

K, (PTU)

= 1

,M,

Km

(rT3)

=0.5

,uM,

and

Vm,>(rT3)

= 60

nmol/mg protein

per

h,

although

theactivityat0.5

_AM

rT3will be -85% inhibited

at 10

,tM PTU,

the activity at 0.5 nM rT3 will be only 1% inhibited

(and only

50%inhibitedat 1 mM

PTU).

Conclusions

regarding

enzymeidentificationbased

solely

onPTUinhibition

aretherefore dangerous since even a single enzyme's

suscep-tibility

to inhibitionvaries withexperimental conditions

(e.g.,

substrate

_{concentration). (b)}

Inmostpublished studies,

partic-ularly

those

dealing

with the brain and pituitary deiodinases (4,

5), high

DTT concentrations (e.g., 20-100 mM)have been used which

might

beexpectedto cause asubstantial

lessening

of PTU inhibition since PTU andDTT arecompetitive

(Fig.

3 B).(c)Furthermore, underconditionswherePTU is inhibi-tory, the inhibited enzyme would display much lower Km's and

V,,,,'s (depending

onPTUconcentrations) becauseofthe

uncompetitive

nature of the inhibition with respect to the

iodothyronine

substrate

(uncompetitive

inhibition being char-acterized

by

aproportional decreaseof

Km

and

Vm.x

with

Km/

V,,.

remaining

constant). A critical analysis of the kinetic data is therefore imperative in ascertaining whether what is

being

measuredis the residual activityof thehigh

Km

enzyme

at low substrate concentrations in an environment of

high

B

- DHL

o DTT

Figure 8. Thiol activation of5'-monodeiodinationof

GSH rT3

by

renalmicrosomes.Theincubationconditions were as described in Fig. 1, except that the incubation , timewas3 min. Each point is the mean of three 3 2 1 determinations, each done in duplicate, from three

differentmicrosomal preparations.

TableII.Kinetic Characteristics of Renal Microsomal Iodothyronine5'-Deiodinaseat Low (Nanomolar) and High (Micromolar) Substrate Concentrations

Low[S] High

_[S]

0.1-10nM 0.1-10)M

Response to GSH ++ 0

(1-5mM)

Inhibition by

PTU Ki= 100MM KiJ=1 tM

(Noncompetitive) (Uncompetitive) lopanoate K,=0.9 mM K.=9.21M

(Competitive) (Competitive)

Dicoumarol Minimal* Ki=7.5_,M

(Competitive)

Mechanism Sequential Ping-pong

(ordered)

Km

rT3 2.5nM* 0.5 IM

T4 0.63nMt 5 ,M

rT3 16pmolt 60 nmol

T4 0.58pmolt 2.1 nmol

Temperature Concave Concave upwards

activationcurve downwards

*Assayedat50AMPTUand 5 mM DTT; Assayed at100uM PTU and 2.5 mMDDT; §mgprotein'hV'.

PTUand DTT,orthe activity ofadistinctly differentlow

Km

enzyme.

Several distinctive features emerge from analysis ofthe kineticdataobtained with the kidneyenzyme atlow substrate

concentrations:

(a) Although the

rT3-deiodinating

activity at low

_[SJ

differed only modestly from that projected from the Km and V,, obtained at

high

substrate concentrations,

T4-deiodinating

activityat low

[SI

was

appreciably

higher than that extrapolated from the kinetic parameters derived from studiesat

high

[SI

and, incontrast tothe observationsat

high

[S],

the enzymeshowedamuch lowerKmfor

T4

than for

rT3.

Suchareversal of substrate

preference

for

T4

than forrT3 has also been

reported

with the type II enzyme of the brain and

pituitary;

but unlike the brain and

pituitary

type II enzyme, where the V

_I,,Km

ratio forT4 was

high

and where the

rT3

deiodination

washalf-maximallyinhibitedbyvery low(2

nM)

concentrations

of T4

(5),

the

kidney

enzyme atlow substrate

concentrations

showedamuch smaller

V.t

for

T4

and

required

muchhigher

concentrations

of T4

(200 nM)

forhalf-maximal inhibition of rT3 deiodination

(data

not shown). The reason

for this limited

ability

of

T4

to suppress

rT3

deiodination,

despitethe lower Km for

T4

than for

rT3,

isnotentirelyclear. Itshould be noted, however,that inatwo-substrate

sequential

mechanism,

the

K,

ofanalternate substrate

(e.g., T4

inrespect torT3)isnot a truedissociationconstant, but isa

steady-state

constant (23)thatcould assumea high value when the rate of degradation oftheternary complex formedby thecompetitive inhibitor is slow, asappearsto be the case with T4relativeto rT3 in the kidney microsomes. (b) At 5 mM DTT, 1 mM PTU surprisingly inhibited the enzyme at low

[S]

by >90% (Fig.2), a levelof inhibitionthat wasconsiderably higherthan that calculated from the kinetic parameters at high

[S];

this suggests the presence of a separate

PTU-sensitive,

low

Km

enzyme.The renal low

Km

enzymethusappearstodifferfrom the type II enzyme described in the cerebral cortex and pituitary (4, 5) in being moderately sensitiveto inhibition by PTU. (c) The PTUinhibition at low

[S]

wasnoncompetitive with a

K,

considerably larger(100 AM)than the

Ki

(1

AM) for the uncompetitive inhibition observed at high

[5].

(d)

Iopa-noate,acompetitive inhibitorof rT3 deiodination

(15),

was a much weaker inhibitorat low than at high

[S].

If the high

Km

enzyme werethesolerenal microsomal outer-ring deiodinase, one would have expected, given the nature of competitive inhibitory kinetics, that a fixedconcentration of the inhibitor would be more,rather thanlesseffective at low

[S].

Thus the observation thatthe

Ki

of iopanoate is-

100

_{times greater}at nanomolarthan at micromolar

[S]

isadditional evidence for the presence ofaseparatelow

Km

deiodinase, lesssensitive to iopanoate because of its higher affinity for the substrate. Dicoumarol,apotent competitive inhibitorat high

[S],

was a weak inhibitor at low [S]; the structural unrelatedness of dicoumarol and

iodothyronine

raises the possibilitythat dicou-marol could be amodifier interacting withaseparate binding siteon thehighKm enzyme alone,perhaps

assumingcompetitive

inhibitory kineticsas aK-system effector (24).

To eliminate the activity ofthe

conventional

high

Km,

PTU-sensitive

renal microsomal

5'-iodothyronine

deiodinase, we added PTU(50-100

,uM)

in almost allexperimentsat low substrate

concentrations

(except for the

experiments

in

Figs.

1, A and B, 2, and 8). Actually, the degree of inhibition by PTU ofa true high Km enzyme

operating

at

subnanomolar

substrate

concentrations

is

negligible,

for reasons

explained

above. Nevertheless, the

possibility

that the

properties

ofthe low Km enzyme arereally those ofa

PTU-modified

high Km

enzyme needstobe

considered.

Wethink it

unlikely

thatthis is thecase because(a)aPTU-modifiedenzymewould

probably

not be PTU

sensitive,

as the low

K.

enzyme appears to

be;

(b)

PTU-

modification

of the

high

Km

enzyme would be unlikely to alter

iopanoate binding

without

similarly altering

substrate

binding;

and,

(c)

the difference between

predicted

andobserved reaction rates

(Fig.

1,

A and

B)

atlowsubstrate

concentrations

were obtainedinthe absenceof

PTU.

Reaction mechanisms. The

essential

feature ofthe

ping-pong

mechanism

is that the enzyme reacts with one ofthe substrates toformaMichaelis

complex

that is

followed

by

the dissociation of the first

product

leaving

a modified enzyme form usually

containing

a

moiety

ofthe first substrate. The second substrate interacts withthemodifiedenzyme

only

after

dissociation

ofthe first

product.

Basedonkineticdataobtained

at high substrate

concentrations,

a model has been

_proposed

forthehepaticand renalhighKmenzymein whichanoxidized form of theenzyme(possiblyasulfenyl

iodide)

is first generated during substrate conversion,and reducediodothyronine product is released; DTT is involved only in the reconversion of the oxidized enzyme to the native form with release ofiodide:

DT1

E +rT3 EErT3 - T2+

EO,*I

- _{E + I- +}

_DTTO,

(1) (2) (3)

(4),T

Eio.

PTU +1-.

The ping-pong reaction mechanism is

usually diagnosed

by findingparalleldouble reciprocal plotsof initialrate data and has been experimentally verified for the

kidney

and liver enzyme in a number oflaboratories (10, 11),

including

our own(3).It mustbe

emphasized, however,

asCleland hasdone (25,26), that theparallel initial velocitypatterns in ping-pong mechanisms are observed only when the modified enzyme form generated after the release of the first product

(in

this case

EO,

*I) is

kinetically

stable

during

the time

period

ofthe reaction. At very low concentrations of the iodothyronine substrate and high concentrations of DTT, this may not be the case, since the rate offormation ofthe enzyme-substrate complex might conceivably be small as compared with its degradationandrapid

equilibrium

conditions

might

not prevail. Furthermore, if

_E.,*

I is the

intermediate

enzyme form and themechanism ofPTUinhibition is by formation ofamixed disulfide with

_E.,*

I [as

envisaged

by Leonard andRosenberg (27) and byVisseret al.(28)],then inpresence of excess PTU, the reaction will be diverted towards the formation of the PTU-complex (step4) and theapparentfirst-orderrateconstant forthereactionof enzyme andsubstratetogive iodide willbe nearly zero

(ignoring

the minute amount of iodide generated duringthe first catalytic cycle). RaisingDTTlevels will divert the reaction flux from the dead-end formation of the PTU-complex (step 4) (or the spontaneous hydrolysis ofthe

_E..

* I complex)to

thiol-dependent

production of iodide(step 3) and thus increase the apparent rate of iodide generation (without

affecting

the true rateof combination betweenEandS).This would decrease the slopeof thereciprocal

plot5

and intersecting rather than parallel lines would be observed on Lineweaver-Burk plots, even if the reaction mechanism isbasically ping-pong. This becomes particularly relevant at low substrate concentrations wherethePTU-inhibitionissmall and measur-ablereactionrates can beobtainedeven atfairly high concen-trations _{of PTU. Considerable caution must therefore} be exercised in interpretingtheintersecting Lineweaver-Burk plots at very low iodothyronine concentrations (as observed in the

5The

lack of slope effect in double reciprocal plots of ping-pong mechanisms is due to a constancy of the ratio of free enzyme [E] to the central complex (F), irrespective of the concentration of the second

substrate, because of a lack of reversible connection between E and F

(23).

present studiesand in those ofLarsenand co-workers

_[4-6]),

asevidencefora

separate

(sequential)

kinetic mechanismand,

therefore,

fora

_separate

enzyme.

Whether the reaction mechanisms observed at

high

and lowsubstrate concentrationsaredifferentor not,other mani-festationsof the

enzymatic activity

atlow substrate

concentra-tions, notably

the differential response to

GSH,

differential response to both

uncompetitive

and

_competitive

inhibitors anddissimilar Arrhenius

displays

are,

indeed, suggestive

of the presence ofa_separatelow

Km

enzyme inrat

kidney

microsomes. Such a low

Km

_enzyme could be

quantitatively

important

at

physiological

concentrationsof

circulating T4

and could make

a

_significant

contributiontothe

T3 production

rate.An

estimate,

based on our

_{experimentally}

observed renal and

hepatic

mi-crosomal

_yields

inrats

_(assuming

similar kinetic

properties

for the enzyme activities in these two

_organs),

indicates that at

circulating

levelsof free

_{T4 [3}

X

10-"1

M

(29)],

-38and 80

pmol

of

T3/100

g

body

wt could be

_{generated daily by}

the

high

and low

Km

enzymes,

respectively,

in these two organs,

thus

_accounting

for

approximately

half of the total

daily

production

rateof

T3

in_{rats, estimated}

_by

AbramsandLarsen

(30)

tobe -250

pmol/100 g/body

wtd. The

findings

ofLum

etal.

₍₃₁₎

that theserum

T3

autoregulatory

systemin

_euthyroid

humans with low

circulating T4

concentrations is

_minimally

affected

by

PTU

and

iopanoate

might

be

explained

on the basis ofthe relative

insensitivity

ofthe low

Km

enzyme to

thesetwoinhibitors.Therelative

_{insensitivity}

toPTU and the

partial inhibitory

kinetics observed with this inhibitor when enzymeactivitiesaremeasuredatlowsubstrateconcentrations may alsoaccount for theobservations ofSilva et al.

(6)

that,

in PTU-treated rats,

circulating T3

concentrations cannot be lowered below aboutone-third of the normallevels (evenwith

a

dosage

as

_high

as ₁

_mg/

100g

body wt).

Acknowledgment

This workwassupportedbygrant A-2585 from the National Institute ofArthritis, Diabetes, DigestiveandKidneyDiseases.

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