Biological role of carbamoyl pyridoxal 5′-phosphate

0 Acad6mie des sciences Biochitie / Biocb~mistry

/ Elsevier,

Paris

Biological role of carbamoyl !&phosphate

pyridoxal

R61e biologique du carbamoyl pyridoxal Y-phosphate LUCIA TERZUOLI

‘, MARIA PIZZICHINI I, ROBERTO PAGANI I, ROBERTO FABIO PONTICELLI 2, ROBERTO LEONCINI ‘, ENRICO MARINELLO I*

’ Istituto 2 kituto

di Biocbimica e di Enzimokyia, di Chimica Organica, Facolta

Facolta di Medicina e Cbirurgia; di Farmacia, Universita de Siena, Pian dei Mantel&i

GUERRANTI

44, 53100

Siena, II&

Nous avons synthetise un nouveau combine, le carbamoyl pyridoxal5’-phosphate, par condensation du PLP avec le KCNO. Nous avons demontre qu’il peut etre obtenu en conditions physiologiques de pH, temperature et de concentrations des react&. Formation et degradation du C-PLP sont des reactions facilement reversibles ; elles ont lieu par un mtcanisme chimique, non enzymatique, au moins dans les tissus de rat. Toutefois, differentes considerations conduisent a la conclusion que le combine, sa synthbe et sa degradation exercent un role biologique dans la cellule. 11represente une (( synthese protective D, une (( reserve variable t) du PLP et du KCNO, qui peuvent Ctre capttes par de nombreuses proteines, enzymes et metabolites ; de cette man&e il regule beaucoup de fonctions metaboliques de la cellule.

Mats-cl&

: carbamoyl

pyridoxa/5’-phosphate,

r6le biologique

ABSTRACT A new compound, carbamoyl-pyridoxul5 ‘-phosphate (C-PLP), was synthetized by condensation of pyridoxal5 ‘-phosphate (PLP) with KCNO. It may be obtained under certain physiological conditions ofpH, temperature and concentration of reagents. Formation and degradation of C-PLP are readily reversible chemical reactions, not involving enzymes, at least in rat tissues. However, different considerations suggest that synthesis and breakdown of C-PLPpkry a biological role in the cell, providing ;Orotective synthesis’ and a ‘variable reservoir’ of PLP and KCNO, which can be trapped by other proteins, apoenzymes and metabolites, to regulate many cell metabolicfinctions.

Keywords:

carbamoyl

pyridoxal

T-phosphate,

Note pr&nt& par Jean Rosa Note remise le 25 novembre 1996, accept& apt&s r&&ion *Correspondence

biological

le 21 avril 1997

and reprints

C. R. Acod. Sci. Paris, Sciences 1997. 320.435-440

role

de la vie / Life Sciences

‘,

L. Tetzuoli

VERSION

et

al. ABRGGGE

Nous avow demon& la synthese d’un nouveau compose, le carbamoyl pyridoxal 5’-phosphate (C-PLP) en Ctudiant I’inhibition d’une enzyme, la L-threonine dtaminase, par l’action du carbamoyl phosphate ou du KCNO. L’inhibition observee a ete attribute a une combinaison du CP on du c KCNO avec le PLP lit a l’oloenzyme, ou avant son association avec I’apoenzyme. Nous avons obtenu des risultats analogues avec une autre enzyme, la transaminase glutamique-pyruvyque, qui est aussi PLP-dependante. Par la suite nous avons demontre directement que le carbamoyl pyridoxal 5’-phosphate (C-PLP) peut Ctre forme chimiquement par condensation du carbamoyl phosphate ou du KCNO avec le pyridoxal 5’-phosphate. La structure du C-PLP a ete demontree avec les criteres suivants : analyse spectrale (dans le visible et l’ultraviolet) ; analyse a I’NMR et a l’infrarouge ; spectrometrie de masse. Par cette recherche, nous avons essay6 d’etablir si le carbamoyl 5’-phosphate a un role biologique, et precisement s’il peut Ctre forme en conditions physiologiques de pH, de temperature et de concentrations ; s’il peut &tre forme ou dtcompose en presence des extraits de tissu, et s’il explique une quelconque fonction dans le metabolisme cellulaire. Pour ces raisons, nous avons ttudie la formation et la decomposition du carbamoyl pyridoxal 5’-phosphate a basse concentrations des reactifs (PLP et KCNO), a pH physiologique (compris entre 5 et 8,5), B la temperature de 37 “C. Les concentrations (finales) des rtactifs ttaient 0,5 mM PLP, 0,5 mM KCNO, 0,25 mM C-PLP. La formation et la degradation du C-PLP ont Ctt determinCes par spectrometre, en enregistrant le spectre et les variations de I’extinction du PLP (ES& et du C-PLP (E&. La formation du C-PLP a ete controlee aussi avec 1’HPLC (en chromatographie liquide a haute resolution), selon la proddure que nous avons preddemment decrite, utilisant comme phase mobile = 0,Ol M potassium-phosphate (pH 5) + 2 % methanol, vitesse de sortie (ecoulement) = 1 mL/mL. DPtection a 254 nm. L’analyse quantitative dards adtquats.

a ete conduite

en utilisant

des stan-

Nous avons obtenu les resultats suivants :

Introduction In a previous study [ 1 ] we showed that a new adduct, the carbamoyl pyridoxal 5’-phosphate (C-PLP), can be synthesized by condensation of PLP with either carbamoyl phosphate (CP) or KCNO. The compound was synthesized and identified on the basis of element analysis and spectral data, and its IUPAC name is 3,4-dihydro-2Hpyrido [3, 4-e] 1,3-oxazin-2-one. The fact that the com-

i) La formation du C-PLP, a partir de PLP et de KCNO, a lieu a tous les pH consider& ; le pH optimal est compris entre 6 et 7. ii) La decomposition du C-PLP a lieu au m&me intervalle de pH, avec valeurs maximales entre pH 6 et 7,5. iii) La reaction est irreversible, avec une valeur de kc9 = 1,30 t pH 7,0,43 a pH 7,5 B 37 “C. iv) En presence d’extrait de foie (et des autres tissus de rat) ces valeurs ne presentent aucune modification : cela demontre que la formation et la degradation du C-PLP n’ont pas lieu enzymatiquement, du moins dam les organes du rat. Le plus important de nos resultats est le fait que le C-PLP peut etre forme et decompose facilement en conditions physiologiques de pH, temperature et a basses concentrations des reactifs, de I’ordre qui ont ete d&rites dans la cellule. Par consequent, la reaction peut avoir lieu dans la cellule, in vivo, dans les deux directions. Nous devons considerer que, in vivo, les concentrations du PLP, du KCNO et du C-PLP ne sont pas &glees seulement par la constante d’equilibre que nous avons calculee in vitro, mais par beaucoup d’autres facteurs. Nous devons tenir compte du fait que le PLP libre est en equilibre continu avec le PLP lie aux apoenzymes, dont il est la coenzyme essentielle ; en outre le PLP libre peut inhiber beaucoup d’activites enzymatiques et de proteines : le KNCO peut etre forme par decomposition du carbamoyl phosphate (un metabolite qui est forme par condensation, pour l’action de la carbamoyl phosphate synthetase I et II), et peut-Ctre deplace par beaucoup de metabolites, des enzymes et de proteines, sur lesquelles il explique une activite inhibitrite (par action avec les groupes -NH,). 11 est evident, par consequent, que i) les concentrations cellulaires du PLP, du KCNO, du C-PLP, sont soumises continuellement a des variations qui sont ftnement controlees ; ii) elles controlent, a la fois, l’activite de beaucoup d’enzymes, des proteines et les concentrations d’autres metabolites ; iii) la reaction PLP+KCNO+-+C-PLP explique un role particulier dans la vie cellulaire. Nous concluons que la formation du C-PLP peut &tre une (( synthese protective )) et que ce mttabolite peut representer une ccreserve variable )) de PLP et des KCNO qui, a la fois, controlent l’activite de nombreux enzymes et proteines. En conclusion, le C-PLP est un metabolite qui est forme chimiquement, a une importance biologique in vivo et merite surement d’etre ulterieurement etudie.

pound is obtained from two well-known compounds of biological importance, namely PLP and CP, and from another such as KCNO, which is very easily formed by spontaneous decomposition of CP [2], suggest that C-PLP may be an interesting compound not only from a chemical point of view. For this purpose, we investigated here whether C-PLP is readily formed and degraded under physiological conditions. The chemical reaction: C. R. Acad.

Sci. Paris, Sciences

de la vie / Life Sciences 1997. 320,435440

Carbamoyl-pyridoxal PLP + CP (KCNO) was

++ carbamoyl (C-PLP)

demonstrated

to occur

pyridoxal

PI easily

in

under certain physiological conditions concentrations of reagents); through reached the conclusion that reaction biological role.

Materials

k, = rate constant

5’-phosphate buffered

solution,

(pH 7, 37 “C, low a careful analysis, we [l] and C-PLP play a

and methods

PLP, carbamoyl phosphate, gents were obtained from commercially

available

Preparation One

purity.

gram

(3.77

pyridoxal

mmoles)

5’-phosphate

of PLP was

dissolved

in 30 mL

of distilled water, with just enough diluted (0.01 M) NaOH to keep the pH below 7. KCNO was added in the molar ratio KCNO/PLP of 2:l. The mixture was stirred for 15 min, adjusted to pH 4 with concentrated red at 25 “C for 24 h. The bulky precipitate red and washed with distilled weighed 0.81 g (2.64 mmoles: melting point of 198 “C (dec.).

Formation supernatant

and

Formation

of C-PLP

CP can

be used

instead

of KCNO.

The final product 70%) and had The

tical, but slower and the ratio CP/PLP the purposes of this study we carried synthesis of C-PLP using KCNO.

reaction

assay

and

Male Wistar 25 “C) were

preparation

of enzyme

rats, weighing fed with pellets

water ad libitum. 10% homogenates 50 mM K-phosphate

They of

mixtures

instead

a

is iden-

of 4:l is used. For out the chemical

fractions

were killed by decapitation, and various tissues were prepared in buffer, using an Ultraturrax appara-

Determination equilibrium We

of rate constants constant of reaction

calculated

k, = rate

constant

separately

k, and

k, for

6.66

mM

supernatant,

were

run.

of C-PLP

of PLP and

as described and treated

C-PLP

formation of the same disappearance of the of c-PLP

band, band

indicating release of PLP. The was an index of the synthesis

““\, CH/OCONH2 I H,OPO,H,

[l 1.

of C-PLP

de la vie / Life Sciences

PLP,

30 and 60 min at 37 “C. Blanks were run above. The denatured proteins were removed also as described above.

A

a second-order reaction of the type k, is the slope of the straight line l/A versus t. The assay mixtures mM PLP and 0.1 mM K,HPO, bufof 50 mM KCNO was added. Incuat 37 “C for 15 min.

C. R. Acad. Sci. Paris, Sciences 1997. 320,435440

of rat liver

the

reaction

mM

were diluted using 50 mM PLP concentration of 0.5 mM. water was added to the assay

HO

k, and

for synthesis

Formation of C-PLP is [A12>B. The constant obtained by plotting (1 mL) contained 0.25 fer (pH 5.5-8.5); 10 pl bation was carried out

k, and

mL of Norit

1.33

PLP was determined spectrophotometrically by its specific band at 380 nm and by its specific extinction coefficient, E,,,. Decomposition of C-PLP was followed by the

200-250 g (housed at 23from Piccioni (Brescia) and

(50%, v/v) for 15 min (1.5 10 mL of clear supernatant).

contained

in rat liver

The assay mixtures contained 1.33 mM C-PLP, 50 mM K2HP04 buffer (pH 7.5) and supernatant of 50 mg liver, in a final volume of 1.5 mL. A quantity of 2 M HCI was added (0.5 M final concentration) at time 0 and then after

tus (IKA Werk). The homogenates were centrifuged at 260 000 xg for 1 h in a Beckman L65 ultracentrifuge with a type 7O.ti rotor. The supernatant was adjusted to the original volume with the same buffer, and treated with a Norit suspension suspension for

of C-PLP

and the clear supernatants K,HPO, (pH 7.5) to a final Blanks, in which distilled

Determination Animals

decomposition

mixtures

Decomposition

HCI and stowas then filte-

water. recovery

of C-PLP

KCNO and supernatant of 50 mg of fresh liver in 0.05 M buffer (pH 7.5) to a final volume of 1.5 mL. A quantity of 2 M HCI was added (0.5 M final concentration) at time 0 and then after 30 and 60 min at 37 “C. The denatured proteins were removed by centrifugation at 8 000 xg (15 min)

KCNO, and all the other reaMerck and were of the highest

of carbamoyl

for dissociation

in the cell

Dissociation of C-PLP is a first-order reaction. The constant k, is the slope of the straight line obtained by plotting in In[Al versus t. The assay mixtures contained 0.25 mM C-PLP and 0.1 mM buffer (pH 5.5-8.5). Incubation was carried out at 37 “C for 15 min.

The

Materials

5phosphate

Figure

1. The structure

of C-PM

L. Terzuoli

et al.

C-PLP

was

also

determined

spectrophotometrically

The

reversibility

of the

the determination (table I).

Analysis performed

i) formation and decomposition 37 “C, at all pHs between 5.5

The

of synthesis and degradation products was also by separating PLP and C-PLP by HPLC (high

performance reported [3].

A

liquid chromatography) Beckmann HPLC

as

previously equipped

apparatus,

with two 11 OB pumps and a mod. used. The mobile phase consisted phosphate buffer (pH 5.0) and 2%

ii) the 7.0;

166 UV analyzer were of 0.01 M potassium methanol at a flow-rate

results

of

reaction

through its specific extinction coefficient at 275 nm, E2,s. Its degradation was followed by the decrease in the same band, its formation by the increase in E,,,.

can

optimal

at

be summarized

pH

iii) the optimal and 7.5;

k,/k,

for

pH

integrated same

through temperature

as follows: of C-PLP and 8.0;

synthesis

for

was the

was

degradation

both

between was

occurred pH

6.8

between

pH

of 1 mUmin. Detection was performed at 254 nm. When separated by HPLC, C-PLP and PLP showed remarkably different retention times (C-PLP = 9.90 min; PLP = 11.37 min). They were detected in the eluted fractions through their UV extinction. Quantitative analysis was carried table

out by comparing extinction coefficients standards. No other compounds were

during

the degradation

at and 7.0

A

with suidetected

experiments.

Equipment The

spectra

recorded photometer.

and using

the

specific

extinction

a computerized

coefficients

Beckman

were

DU8

spectro-

Calculations

Calculation of k values was performed 386 IBM-compatible computer and gramme. The specific molar extinction for calculations were:

using an Olidata the Enzfitter procoefficients used

nm

B

E,,j for C-PLP = 5.08 mmol-’ E,,, for PLP = 4.8 mmol-’

x cm-’ x cm-’

A

1,4

1

12

Results Figure

1

2 shows

KCNO,

and

the

formation

of C-PLP

its decomposition

from

PLP

0.8

and

at 37 “C.

Both reactions were followed E 27s and E,,, (see Materials and

0.6

through methods).

the

variations

in 094

072 Table PLR

I. Values

of rate

constants

for the

reaction

KCNO+PLP

tf

0 k, (mar’

PH

5-I)

k eq

k, (s-‘l

5.5

5.83

. lo-*

- 1.39

10m3

6 6.8

2.21 6.90. 5.90

. 10-l lo-* 1 o-2

- 9.23 - 2.30. - 4.40

. 10-3 IO-* 1 o-2

2.40

lo-*

- 5.52

1O-2

1.30 0.43

no reaction

- 3.93

. 10-l

0.00

7 7.5 8.5

k,: formation C-PLP

into

perature, in Materials

438

C-

of C-PLP, KCNO

37 “C. and

and

from

KCNO

PLP;

k,,:

The composition methods.

and PLP; equilibrium of the

Figure

23.90 3.00

mixtures

(k,/k,).

2. Formation

anddecomposition

A. C-PLP formed through (- --- -i on/y PLP; ( --) recording EjaO and E,,, mal volume PLI?

k,: decomposition constant

assay

nm

41.90

of Tem-

is reported

(10

pL)

B. Decomposition tion. The complete Materials and spectrophotometer

of 50 mM

solution

of C-PLP (0.25 composition

methods.

C. R. Acad.

of C-PLP

at 37

“C, pH

7.

chemical reaction between KCNO and PLP PLP + KCNO. The reaction was followed every 5 min for 15’ after addition of a miniof KCNO

mM solution) of the assay

during mixtures

Spectra were recorded at 37 “C. Values are final Sci.

Paris,

Sciences

to I mL of0.25 15’ofincuba is reported

with a Beckman concentrations. de

mM

in DU8

la vie / Life Sciences 1997.320.435-440

Carbamoyl-pyridoxal5’-phosphate iv) at pH

8.5,

no synthesis

but

only

degradation

of C-PLP

ues of k,.

occurred; v) the

so that reaction

prevailing Table

was

perfectly

at lower II shows

pH that

and both

reversible

at pH

degradation

above

the

formation

and

7.

degradation

of C-PLP were the same, irrespective of the presence or absence of tissue extracts. Similar results were obtained with extracts of other rat organs, but are not shown here for the sake

of brevity.

otides. cytosolic chondria

II. Synthesis

and

decomposition

lncuba tion time at 37 lmin)

of C-PLP

A

in rat liver

B

superna-

c

Cells, therefore, and KCNO that

D

-91

- 82

10

-40

- 40

- 68

-50

-2

-17

II

Discussion Formation

contain finely

concentrations controlled.

degradation

of C-PLP

occur

readily,

at low

trate that the reaction vitro; and since such that the reaction may if C-PLP

in rat tissues, tions needs

does

occurs readily and spontaneously in conditions occur in vivo, it is likely occur in the cell. not

form

and its behavior to be investigated,

which are reported may play a biological

below, role.

Reaction [l] is controlled regulate many other enzymes releasing PLP and CP.

or degrade

enzymatically

in other biological preparaseveral considerations,

also

indicate

that

reaction

nuous zymes.

ent our

equilibrium

that and

the free formed;

This last observations we

has a concentration to proteins [4]. We

of have

obtained positive results using the lowest concentrations of PLP (10 pM) and CP (40 FM), which fall in the range

of

Free PLP is easily that regulate amiand have very low val-

de la vie / Life Sciences

any

proteins

of PLP, CP of free

have demonstrated deaminase (E.C.4.2.1

free

PLP and

until contiholoen-

control each of PLP-depend-

is in [14] .16),

PLP

proteins, which low k, of PLP for

KCNO mutually and regulation

conclusion [I 41.

121

the formation 5’-phosphate

excess

other the

+

coenzyme to apoenzymes this is ensured by the

agreement

with

the inhibition a pyridoxal

of 5’-

phosphate-dependent enzyme, by carbamoyl phosphate and KCNO. The inhibition was ascribed to a direct effect at the substrate binding site of the holoenzyme, and also to interference with the association reaction: PLP + apoenzyme

the sensibility of the spectrophotometer. substracted from many apoenzymes noacid metabolism inside the cell

metabolites,

C-PLP,

must

C-PLP

concentrations

between PLP, CP and by inhibition

[I]

cell that in vitro, factors, and it can

e

to avoid

with PLP, CP

of reaction

in the different

of intracellular

links are

enzymes. previous

groups, of free

enzymes

synthesis,

In practice, other’s levels,

-NH,

data above indicate that of carbamoyl pyridoxal role, since they represent:

i) a fine control and KCNO;

In fact, L-threonine [II

by complex mechanisms and reactions, trapping

Intracellular liver PLP normally 2 pM and it is not firmly bound

C. R. Acad. Sci. Paris, Sciences 1997.320.435-440

All the reported and decomposition play a biological

apoenzymes holoenzymes

free

the equilibrium

iii) a variable reservoir of PLP for regulates different enzyme activities:

concentration of reagents, under certain physiological conditions of temperature (37 “C) and pH (6.5-7.5). At pH 7 the reaction shows a k,, of 1.30. Our data demons-

Even

with

are

< 18pM

121: approximately at pH 7.15-8.86

reacts readily on proteins.

proteins

ii) a protective or CP/KCNO;

and conclusions

and

of CP splitting per minute

A\

apoenzymes

impornucle-

[12],

PLP + CP (KCNO)

I = synthesis of C-PLP; II = degradation of C-PLP. The composition of the assay mixtures and all details are reported in Materials and methods. I: A and B contained PLP and KCNO, C and D contained PLP and CP. Only B and D contained 1.25% rat liver supernatant. The results are expressed as decrease in PLP (E,& or formation of C-PLP (E,,,), which were equivalent. II: A and B contained C-PLP, C and D contained PLP alone. Only B and D contained 1.25% rat liver supernatant. The results are expressed as a percentage decrease in C-PLP for A and B and as a percentage decrease in PLP for C and D.

biological pyrimidine

13]:6nmol/gwetwt. 1131.

As a consequence,

10

proteins,

by mitochondrial synthetase I and II [8, 91 and can diffuse from mito[lo, 111. intracellular concentrations

be much more complicated because it depends on many be represented as follows:

I

on many [5-71.

(CP) is of great of urea and

is a product it decomposes KCNO effects

effect

be eliminated

phosphate biosynthesis

ofCParelow [12, in liver mitochondria

(37 “C). negative

“C

must

It is formed synthetase to cytosol

KCNO 1.5% of Table tant.

has a negative

excess

Carbamoyl tance in the

7, synthesis pH

PLP also

any

in the cell

Since

carbamoyl

phosphate

tf

holoenzyme decomposes

spontane-

ously [2] to KCNO, the inhibition of reaction [2] was ascribed to the fact that the last compound reacts with the coenzyme to form a new adduct, carbamoyl pyridoxal 5’phosphate (carbamoyl-PLP: C-PLP) 1141. Similar observations were reported for glutamate-pyruvate transaminase (GPT)

[15].

439

L. Terzuoli

et al

Our investigation has been carried out in vitro; the conof reagents that can be used in such an experall the conditions are only comparable and not

centrations iment, and coincident

with

the situation

in vivo.

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C. R. Acad.

Sci. Paris, Sciences

de la vie / Life Sciences 1997.320.435-440