Evidence that Fe2+ complexes of 3-aminopicolinate and 3-mercaptopicolinate activate and inhibit phosphoenolpyruvate carboxykinase

Vol. 90, No. 3, 1979 October

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 741-749

12, 1979

EVIDENCE THAT Fe2+ COMPLEXES OF 3-AMINOPICOLINATE

AND INHIBIT PHOSPHOENOLPYRUVATE CARBOXYKINASE

ACTIVATE

J. MacDonald

Michael

Institute University

Received

August

AND 3-MERCAPTOPICOLINATE

for Enzyme Research of Wisconsin, Madison 53706

6,1979

SUMMARY: 3-Mercaptopicolinic acid is known to be an inhibitor of phosphoenolpyruvate carboxykinase and 3-aminopicolinic acid permits Fe2+ to activate the enzyme. The potency of mercaptopicolinate is increased by incubating the enzyme with Fe2+ prior to assaying for activity. In the present work, the average combining ratio of either pyridine carboxylate with Fe2+ at pH 7.5 was determined to be 2:l when measured by the method of continuous variation of Job or by elemental analysis of the isolated pyridine carboxylate-Fe2+ complexes. The ratio of 3-mercaptopicolinate or 3-aminooicolinate to Fe2+ that caused the greatest inhibition or activation of purified phosphoenolpyruvate carboxykinase was 2:l. In the absence of Fe2+, neither pyridine carboxylate altered the activity of the enzyme. These resu ts indicate that the two pyridine carboxylates can interact with phosphoeno pyruvate carboxykinase as Fe2+ coordination complexes. INTRODUCTION: acid

are

Quinolinic

structurally

enolpyruvate

acid, similar

effecters

carboxykinase

(E.C.

enzyme --in vitro

(l-9),

inhibit

10) or

slices

(11)

in liver

3-Aminopicolinate boxykinase hepatic

protein

synthesis Lardy cause

Fe2+ enhances

ABBREVIATIONS: p'-disulfonic acid.

it

it

is

complexes

likely with

a model

Fe 2+ .

Others

liver

(1,5,

phosphoenolpyruvate

car-

for

the naturally-occurring increases

glucose

(8,13,14).

--et al.

agents

the

(11,12).

of the carboxykinase these

inhibit

animals

animals

and McDaniel

that

perfused

3-Aminopicolinate

in intact

inhibition

enzyme phospho-

in intact

purified is

and 3-aminopicolinic

two compounds

in the

hypoglycemia

(6).

(1,2,7-g)

the

The first

Fe2+ to activate

hyperglycemia

acid

gluconeogeneic

synthesis

and cause

named ferroactivator

and coworkers

coordination

4.1.32).

respect

nit

of the

glucose

and in this

and causes

mercaptopicolinate ing

permits

(7,B)

3-mercaptopicol

(3)

maintain

by quinolinate

inhibit

beand 3-

the enzyme by form-

have postulated

Ferrozine, 3-(2-pyridyl)-5,6-diphenyl-1, acid. Hepes, 4-(2-hydroxyethyl)-1-piperazine

that

that

3-mercapto-

2, 4-triazine-p ethanesulfonjc

0006-291X/79/190741-09$01.00/0 741

Copyright All right3

@ 1979

by Academic Press. Inc. in anyform reserved

of reproduction

Vol. 90, No. 3, 1979

picolinate (5).

BIOCHEMICAL

inhibits

Because

teract

with

ion.

Whether

the carboxykinase

3-aminopicolinate

carboxykinase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by withdrawing

is without

effect

of transition

metal

carboxykinase

by forming

a complex

3-mercaptopicolinate

ty of phosphoenolpyruvate coordination

complexes

EXPERIMENTAL

PROCEDURES.

ions

(7,8),

with

has been

the enzyme

by interacting

it

too may in-

a transition

and 3-aminopicolinate

carboxykinase

from

on phosphoenolpyruvate

in the absence the

a metal

metal

can alter with

the activi-

the enzyme as Fe

2+

investigated.

Materials. 3-Aminopicolinic acid hydrochloride and 3-mercaptopicolinic acid were gifts of Theodore Resnick and Dr. Harry Saunders, respectively, both of Smith Kline Corp., Phil., Pa. The purity of these agents was established by thin layer chromatography, nuclear magnetic resonance spectroscopy and their carbon and nitrogen contents. The chloride content of the 3-aminopicolinate hydrochloride was determined in order to estimate its formula weight. One mole of 3-aminopicolinate was contained in 180 grams of its hydrochloride. The molecular weight of 3-mercaptopicolinic acid was taken as 155. Ferrozine was from Aldrich Chemical Co. Phosphoenolpyruvate carboxykinase was purified from rat liver cytosol according to the methods of Ballard and Hanson (15,16) with modifications (17). The specific enzyme activity of the preparation used was 9 umoles product x min-1 mg protein-l when assayed at 25°C in the absence of effecters and in the direction of phosphoenolp ruvate formation (represented as 100% enzyme activity in Figures 2, 3 and 4 J . Analyses. Determinations of absorption spectra of solutions of Fe" and pyridine carboxylates were made with a DW-2 UV/VIS Spectrophotometer (American Iron was determined by atomic abInstrument Co.) using the split beam mode. sorption spectroscopy and chloride with kit No. 830-T from Sigma. Total organic carbon was determined with a Dohrmann DC-50 carbon analyzer (18) and total Kjeldahl nitrogen by an ultra micro method (19) by the Water Chemistry Division, Wisconsin State Laboratory of Hygiene. Enzyme assay. Phosphoenolpyruvate carboxykinase activity was assayed in the direction of phosphoenolpyruvate formation as previously described (6-9,17). Prior to assaying for activity, 2.0 to 3.0 ug of purified enzyme were incubated for 10 minutes with or without pyridine carboxylates or FeC12 in a final volume of 0.2 ml of 5 mM Hepes buffer pH 7.5 and 1 mM dithiothreitol. One tenth milliliter of the incubation mixture was added to 0.9 ml of the enzyme reaction mixture to start the enzyme reaction. RESULTS Combining

ratios.

3-aminopicolinate the visible with

Fe'+ are

region.

colored

orange,

complexes

with

and have absorption

The colors

3-mercaptopicolinate,

aminopicolinate,

coordination

dark 466 nm.

and absorption blue-green, The iron

742

3-mercaptopicolinate maxima

at wavelengths

maxima at pH 7.5 a broad in

peak at 625;

the complexes

with

in water

and in are:

and with 3-mercapto-

3-

BIOCHEMICAL

Vol. 90, No. 3, 1979

picolinate

and with

is mixed

with

ing

agent,

solution

3-aminopicolinate

3-aminopicolinate

has an absorption is

over

two to three

ting

that

captopicolinate, green

picolinate.

This

tometric

pyridine

combining

of

carboxylate

were

fast

the color

coordinated

of the

to follow

indica-

3-amino-

an excess

within

seen when is

with

with

changes

Fe(II1)

that

with

of 3-mer-

seconds

to the

FeC12 is mixed

reduced

by the

by conventional

with

3-

3-mercaptospectropho-

formed

the

in water

pH 7.5

(pH adjusted

analyzed

after

for

Fe

2+

with

mixing

carbon,

analysis

of

of the

Fe 2+

nitrogen,

iron

743

five

absorption

readindicat-

1).

indicated with

ratio

3-mercap-

that

the aver-

spectrophotometric

and 3-mercaptopicolinate and pyridine

complexes,

washed

mea-

or with

carboxylate

Precipitates

10 to 20 mM of FeC12 and pyridine

NaOH) were

con-

imnediately

combining

Fe 2t (Fig.

3-aminopicolinate

determined

were

Various

carboxylate

the average

of 3-aminopicolinate

products

with

of pyridine

to one of

concentrations

solubility

The highest

carboxylates,

Fe2'

the sum of Fe 2+ and

(keeping

fraction

was the same as that

by mixing

formed

of Job (20,21).

them to elemental

Fe(I1) complexes

isolated

exceeded

by mixing

with

and the absorbance

carboxylate

and submitting

techniques.

mixed

mole

pyridine

of pyridine

ratio

carboxylates

variation

carboxylate were

0.67

each of the

age combining

pyridine

maxima of the complexes.

around

two molecules

topicolinate

of the

of continuous

constant)

centered

Complexes

were

too

FeC12 and pyridine

at the absorption

were

that

the

ratios

by the method

centrations

is

is

a reduc-

methods.

determined

ing

that

reaction

The average

ings

solution,

Fe(II1)

the

to the color

indicating

which

maximum at 466 nm appears,

color

identical

complex

dithiothreitol,

of FeC13 when mixed

a yellow-red

color

mercaptopicolinate

sured

reduced Solutions

form

If

When FeC13

maximum at 525 nm disappears

as an absorption

the dithiothreitol

Fe(II1).

a magenta-colored

and the absorption

minutes

to Fe(I1).

dark-blue

at pH 7.5,

to orange

RESEARCH COMMUNICATIONS

Fe(I1) and not

is

maximum at 525 nm, is formed. 3+ added to the Fe -3-aminopicolinate

changes

picolinate

AND BIOPHYSICAL

to ten

and chlorine.

times

that

that were

carboxylate with

The colors

water

at and

of the

Vol. 90, No. 3, 1979

BIOCHEMICAL

osi

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-

I

MOLE

I 0.5 PYRIDINE

FRACTION

1: Combining ratios for Fe ", (B) determined by the method of pyridine carboxylates were mixed keeping the sum of the Fez+ and after mixing, the optical density of FeClB plus 3-mercaptopicolinate respectively. The concentration ment with 3-aminopicolinate and

I I 067 075 CARBOXYLATE

Fig.

isolated

complexes

was detected

in

elements

found

complex

indicated

cules

were

that

of pyridine

mixing

marked

the same as those

the the

average

combining

solutions.

of gram atoms

3-aminopicolinate

carboxylate

excesses

in dilute

The number

the complexes. in either

3-mercaptopicolinate (A) and 3-aminopicolinate continuous variation. FeClB and one of the in various ratios in Hepes-NaOH buffer, pH 7.5, the pyridine carboxylate constant. Insnediately was measured. The sum of the concentrations or 3-aminopicolinate were 0.3 and 1.5 mM, of the Hepes buffer was 50 mM in the experi5 mM with 3-mercaptopicolinate.

or the ratios

of either

of the other

three

3-mercaptopicolinate

were

I).

to one of Fe (Table

No chlorine

most

likely

Complexes

FeC12 or of pyridine

two mole-

f ormed

carboxylate

by

had identical

compositions. Enzyme activity.

The method

determine

3-mercaptopicolinate

vate

whether

phosphoenolpyruvate

ous ratios carboxykinase

of

Fe

2+

before

of continuous

and 3-aminopicolinate

carboxykinase

by forming

and 3-mercaptopicolinate assaying

of Fe2+ and 3-mercaptopicolinate

for

of Job was al so used to

variation

were

complexes incubated

enzyme activity (their

sum constant

744

inhibit

(Fig.

with with

2).

and acti-

2+ Fe . the

Vari-

purified

The combinations

at 50 PM or at 100 uM)

Vol.

90,

No.

3, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE Elemental

analysis

Pyridine Carboxylate

Fe complexes 3-mercaptopicolinate

3-aminopicolinate

Probable combining ratiob N:Fe

11.9

+ 0.3

4.0

3-Mercaptopicolinate

11.7

+ 3.6

1.60

aResults for the

are means (? S.D.) of N:Fe of 3-mercaptopicolinate

With either pyridine pyridine carboxylate:Fe ratios of 2:1, 4:l 3-mercaptopicolinate and 3:1, respectively.

175O

0.l I

analyses in

Pyridine

+ 0.3

3-Aminopicolinate

b

of

Ratios in gram atomsa C:Fe

COMMUNICATIONS

I

2+

of of

and

RESEARCH

carboxylate:Fe 2:l 2:l

of 3 different which n = 1.

preparations

except

carboxylate C:Fe ratios of 6:1, 12:l and 18:l indicate ratios of l:l, 2:l and 3:l respectively. N:Fe and 6:l with 3-aminopicolinate and l:l, 2:l and 3:l with indicate pyridine carboxylate:Fe ratios of l:l, 2:l

MOLE 02 I

FRACTION J-MERCAPTWCOLINATE 0.3 0.4 0.5 00 I I I I

_ FsZ+ GOO-0

Fig.

uM)

(ha) 0.7 I

00 I

0.9 I

1.0

-I

2: Effect of continuously varying the ratio of 3-mercaptopicolinate to Fe2+ on the inhibition of phosphoenolpyruvate carboxykinase activity. Various ratios of 3-mercaptopicolinate and FeCl [sum of both effecters equaled 50 pM y) or 100 pM (q )] were incubated wit i; the purified carboxykinase prior to assaying for enzyme activity. The effects of several concentrations of FeCl alone (100 to 0 pM,O ) and of 3-mercaptopicolinate alone (0 to 100 pM, 8 ) are also shown. Each point is the mean of six or more determinations.

74s

Vol. 90, No. 3, 1979

BIOCHEMICAL

MOLE 500°

0.1 I

0.2 I

AND BIOPHYSICAL

FRACTION 0.3 0.4 I I

3-AMINOF’ICOLIN4TE 0.5 Q6 I I

0.7 I

RESEARCH COMMUNICATIONS

(0, n l 0.8 I

a9 I

I.0 I

5300 3 Y c200 E $

loo

iJ 3

SAP

co-IDwAd)

L

1 1.0

Q8

0.6 [Fez+]

Q4 CO, A)

(ml.41

, 0

0.2

Fig.

3: Effect of continuously varying the ratio of 3-aminopicolinate to Fe 2+ on the activation of phosphoenolpyruvate carboxykinase. Various ratios of 3aminopicolinate and FeC12 [sum of both effecters equaled 0.5 mM ( a ) or 1.0 mM ( l )] were incubated with the purified carboxykinase prior to assaying for enzyme activity. The effects of several concentrations of FeC12 alone (1.0 to 0 mM,& and 3-aminopicolinate alone (0 to 1.0 m!l,+) are also shown. Each point is the mean of six or more determinations.

that

caused

ed around average

the a mole

were

activity

while

trations

constant

inhibited trations

carboxykinase

were

center-

3-mercaptopicolinate

indicating

that

the

without

3 shows

the

at 0.5 a 2:l

gave lower or inactivated caused

a slight

inhibit

on the

the effect purified sum

of

the

or 1.0 mM. ratio

in the

absence various

ratios

carboxykinase

before

assaying

3-aminopicolinate

and

the

The peak of activation to Fe in very

the enzyme

as reported

activation

of the enzyme. 746

of

the incu-

previously

high

Fe2+

of 3-mercapto-

of incubating

Fe2+ alone

is

of the carboxykinase.

of 3-aminopicolinate

activations.

In the absence

up to 300 PM in

activity

of the enzyme

the carboxykinase

Fe 2+ .

to one of

effect

the

keeping

around

that

of 3-mercaptopicolinate

and Fe 2+ with

was centered

purified

of the complex(es)

the activity

Fig.

picolinate

or 3:l

ratio

enhanced

picolinate.

1:l

of 0.67

concentrations

mixture

slightly

of the

of 3-mercaptopicolinate

Fe2+,

bation

inhibition

fraction

combining

two molecules added

greatest

of 3-aminofor Fe

2+

enzyme

concen-

of the enzyme 2+

and ratios

of

concentrations (7)

and low concen-

Vol.

90,

No.

BIOCHEMICAL

3, 1979

MOLE 0.5 I

0.4 1501

FRACTION 0.6 I

BIOPHYSICAL

RESEARCH

3-MERCAmOPICOLINATE 0.7 0.6 I I

I I 60 [3-MERCAPTOPlCOLlNATE]

I

01 40

AND

I 60 CpM)

(4

COMMUNICATIONS

4) 0.9 I

1.0 I

I loo Cr, l ) 1 0

L 60 4o

[Fe’+]

C@+O C&t

4: Effect of a strong chelating agent on inhibition of phos hoenolpyruvate carboxykinase by various ratios of 3-mercaptopicolinate to Fe jt . 3-Mercaptopicolinate and Fez+ (the sum of both effecters was kept constant at 100 uM) were incubated with the purified carboxykinase in the presence (& or absence (+) of 1 mM Ferrozine - a strong chelator of Fez+. The effects of Fez+ (60 to 0 pM) on enzyme activity in the presence ( n ) or absence ( l ) of 1 mM Ferrozine are also shown. Conditions were the same as in Figure 2. Each point is the mean of four or more determinations. Fig.

Ferrozine, mixture

a strong

containing

boxykinase

(Fig.

fact

with that

the the

4).

metal

inhibition strong

instead

DISCUSSION:

from

phosphoenolpyruvate

to the incubation

2+ and phosphoenolpyruvate ferrozine

Fe"

agent

interfered

of increasing

the inhibit

car-

(1 mM) prevented

access to the Fe" 2+ of the enzyme by Fe -3 mercaptopicolinate.

does not

with

inhibition

inhibition,

is

the

and interThe by 3-mer-

another

the carboxykinase

indication

by removing

a

enzyme.

The fact

aminopicolinate

act

the

the

was added

the carboxykinase

chelating

3-mercaptopicolinate ion

agent, Fe

By complexing and/or

captopicolinate, that

chelating

3-mercaptopicolinate,

3-mercaptopicolinate fered

metal

that

to Fe"

a specific

causes

maximal

carboxykinase

on the carboxykinase

ratio

as metal-ion

of 3-mercaptopicolinate

inhibition

indicates

that

complexes

747

or activation the for

of purified

two pyridine if

they

or 3-

did

carboxylates not

act

as

Vol. 90, No. 3, 1979

BIOCHEMICAL

complexes

would

their

and would

effects

be independent

The results including picolinate, hibit

unpublished

at positions

in

of these

the signal

are

and ring

mon structure

for

a transition rivatives

is

necessary

the compound

certain

pyridine

of phosphoenolpyruvate

speculate

that

the active

site

the

supported Diabetes

metal

and/or protons

to the ring

the biologically

of pyridine

carboxyl-

by the a-carboxyl derivative

a picolinate 3 of

have on the

Fe *'

carboxykinase

structures

protein, to activate

ACKNOWLEDGEMENTS: support

that

is

a com-

derivative

and

the picolinate

and determines

carboxylates

de-

the action

carboxykinase

and continued by grants

effecters

the enzyme

interest from

is

molecular

(in-

and its

known.

It

interesting

complexes

of the weight

is

or activato

resemble

carboxykinase,

ferroactivators,

such that

(6,24,25).

indebted

to Professor

in the present

the Juvenile

not

as inhibitors

Fe 2+ coordination

of their

or lower

The author

can act is

of naturally-occurring

as ferroactivator permit

will

our

to solutions

of the

bonding

of picolinate

activity

2+

6 than

shows

at position

of bonding

more broadening

probably

between

in-

In addition,

of Fe

causes

study

complex

biologic

(8).

of two molecules

The group

for

is

and 3-amino-

(8).

Why only tors

(22,23).

compounds,

capable

at position

of two molecules

or activation)

potency

quantities

of a transition

a coordination

metal

small

Fe 2+

composed

The bonding

nitrogens

nitrogen

The present

probably

aromatic

derivative

at pH 7.5,

that

increased

or noncompetively

ring

of the proton

compounds.

and one of Fe2'.

hibition

adding

4 and 5, indicating

complexes

group

be a picolinate

that

thirty

to activate

and its

are

2+ ratio.

3-mercaptopicolinate

a compound

group

shows

concentrations

more than

of quinolinate,

must

RESEARCH COMMUNICATIONS

carboxylate:Fe

with

and 3-aminopicolinate

more of a shift

ate

it

NHR work

nitrogen

study

for

a-carboxyl

of quinolinate

active

that

carboxykinase

to Fe2+ by its

as their

of the pyridine

isomers

indicate

the

increase

of a previous

structural

AND BIOPHYSICAL

Diabetes

Association.

748

work.

Henry This

Foundation

A. Lardy

investigation

for

his was

and the American

Vol.

90,

No.

3, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. Z: 10. 11. 12. 13. 14. 15. 16. 17. ii: 20. 21. 2 24. 25.

Veneziale, C. M., Walter, P., Kneer, N., and Lardy, H. A. (1967) Biochemistry 5, 2129-2138. Snoke, R. E., Johnson, J. B. and Lardy, H. A. (1971) Eur. J. Biochem. 24, 342-346. McDaniel, H. G., Reddy, W. J. and Boshell, B. R. (1972) Biochim. Biophys. Acta 276, 543-550. Kostos, V., Di Tullio, N. W., Rush, J., Cieslinski, L., and Saunders, H. L. (1975) Arch. Biochem. Biophys. 171, 459-465. Jomain-Baum, M., Schramm, V. L. and Hanson, R. W. (1976) J. Biol. Chem. 251, 37-44. Bentle, L. A. and Lardy, H. A. (1977) J. Biol, Chem. 252, 1431-1440. MacDonald, M. J. and Lardy, H. A. (1978) J. Biol. Chem. 253, 2300-2307. MacDonald, M. J. and Lardy, H. A. (1978) Bio-organic Chem. 7, 251-262. MacDonald, M. J. (1978) Biochim. Biophys. Acta 526, 293-298. Ray, P. D., Foster, D. 0. and Lardy, H. A. (196rJ. Biol. Chem. 241, 39043908. Di Tullio, N. W., Berkoff, C. E., Blank, B., Kostos, V., Stack, E. J., and Saunders, H. L. (1974) Biochem. J. 138, 387-394. Gullino, P., Winitz, M., Birnbaum, S. M., Cornfield, J., Otey, M. C. and Greenstein, J. P. (1955) Arch. Biochem. Biophys. 5& 252-255. Blank, B., Di Tullio, N. W., Miao, C. K., Owings, F. F., Gleason, J. G. Ross, S. T., Berkoff, C. E., Saunders, H. L., Delarge, J. and Lapiere, C. L. (1974) J. Med. Chem. n, 7065-1071. MacDonald, M. J., Huang, M. T. and Lardy, H. A. (1978) Biochem. J. 176, 495-504. Ballard, F. J. and Hanson, R. W. (1969) J. Biol. Chem. 244, 5625-5630. Phillippidis, H., Hanson, R. W., Aeshef, L., Hopgood, M. F. and Ballard, F. J. (1972) Biochem. J. 126, 1127-1134. Bentle, L. A. and Lardy, H. A. (1976) J. Biol. Chem. 251, 2916-2921. Dobbs, R. A., Wise, R. H. and Dean, R. B. (1967) Anal. Chem. 39, 1255-1258. Jirka, A. M., Carter, M. J., Mary, D. and Fuller, F. D. (1976FEnvironmental Science Technology lo, 1038-1044. Job, P. (1928) Ann. Chem. (Paris) 2, 113. Ingham, K. C. (1975) Anal. Biochem. 68, 660-663. Loiseleur, P. H. (1972) Acta Crystalog. B28, 816-820. Thundathil, R. V., Holt, E. M., Holt, S. L. and Watson, K. J. (1976) J. Chem. Sot. Dalton 1438-1440. Bentle, L. A., Snoke, R. E., and Lardy, H. A. (1976) J. Biol. Chem. E, 2922-2928. MacDonald, M. J., Bentle, L. A. and Lardy, H. A. (1978) J. Biol. Chem. 253, 116-124.

749