Tài liệu Báo cáo khoa học: A kinetic model of the branch-point between the methionine and threonine

A kinetic model of the branch-point between the methionine

and threonine biosynthesis pathways in Arabidopsis thaliana

Gilles Curien, Ste´ phane Ravanel and Renaud Dumas

Laboratoire de Physiologie Cellulaire Ve´ge´tale DRDC/CEA-Grenoble, France

This work proposes a model of the metabolic branch-point

between the methionine and threonine biosynthesis path￾ways in Arabidopsis thaliana which involves kinetic compe￾tition for phosphohomoserine between the allosteric enzyme

threonine synthase and the two-substrate enzyme cysta￾thionine c-synthase. Threonine synthase is activated by

S-adenosylmethionine and inhibited by AMP. Cystathio￾nine c-synthase condenses phosphohomoserine to cysteine

via a ping-pong mechanism. Reactions are irreversible and

inhibited by inorganic phosphate. The modelling procedure

included an examination of the kinetic links, the determin￾ation of the operating conditions in chloroplasts and the

establishment of a computer model using the enzyme rate

equations. To test the model, the branch-point was recon￾stituted with purified enzymes. The computer model showed

a partial agreement with the in vitro results. The model was

subsequently improved and was then found consistent with

fluxpartition in vitro and in vivo. Under near physiological

conditions, S-adenosylmethionine, but not AMP, modulates

the partition of a steady-state fluxof phosphohomoserine.

The computer model indicates a high sensitivity of cysta￾thionine fluxto enzyme and S-adenosylmethionine concen￾trations. Cystathionine fluxis sensitive to modulation of

threonine fluxwhereas the reverse is not true. The cysta￾thionine c-synthase kinetic mechanism favours a low sensi￾tivity of the fluxes to cysteine. Though sensitivity to

inorganic phosphate is low, its concentration conditions the

dynamics of the system. Threonine synthase and cystathio￾nine c-synthase display similar kinetic efficiencies in the

metabolic context considered and are first-order for the

phosphohomoserine substrate. Under these conditions out￾flows are coordinated.

Keywords: allosteric activation; branch-point; kinetic com￾petition; ping-pong; sensitivity coefficient.

Metabolic branch-points display a very large diversity in

terms of the number of the enzymes involved, the kinetic

mechanisms of the competing enzymes and the number as

well as the nature of the allosteric controls. Whether such

diversity in the organization of the branch-points reflects

differences in the branch-point kinetics is not well known.

Indeed, detailed models that take into account the individ￾ual enzyme kinetic properties in their metabolic context are

scarce. Fluxpartition at the dividing point of several

pathways has been studied both theoretically [1–3] and

experimentally [2,4–7]. Some studies used the framework of

metabolic control analysis for this purpose [6,7]. However,

the allosteric controls of the branch-point enzymes are not

taken into account in these experimental studies. Also the

occurrence of branch-point two-substrate enzymes and the

consequence of their kinetic mechanisms for the partition of

fluxin the systems studied previously have not been

considered.

The present paper proposes a computer model of the

branch-point between the methionine and threonine

biosynthesis pathways in Arabidopsis thaliana (Fig. 1). The

computer model was validated in vitro and used to ex amine

the branch-point kinetics in detail and to obtain insights into

the kinetic controls of methionine and threonine synthesis in

plants.

The branch-point between the methionine and threo￾nine biosynthesis pathways (Fig. 1) involves a two-substrate

enzyme (cystathionine c-synthase, CGS) and an allosteric

enzyme (threonine synthase, TS). These enzymes compete

kinetically for their common substrate, phosphohomoserine

(Phser), in chloroplasts [9–11]. CGS catalyses the formation

of cystathionine, the precursor of methionine, by condensa￾tion of Phser and cysteine. The reaction follows a ping-pong

mechanism [12]. In the competing branch, TS catalyses the

formation of threonine from Phser. In plants, TS is sti￾mulated in vitro by S-adenosylmethionine (AdoMet) in an

allosteric manner [10,13–16]. AdoMet is a direct derivative

Correspondence to G. Curien, Laboratoire de Physiologie Cellulaire

Ve´ge´tale DRDC/CEA-Grenoble, 17 rue des Martyrs,

38054 Grenoble Cedex9, France.

Fax : + 33 4 38 78 50 91, Tel.: +33 4 38 78 23 64,

E-mail: [email protected]

Abbreviations: AdoMet, S-adenosylmethionine; CGS, cystathionine

c-synthase; Phser, phosphohomoserine; TS, threonine synthase.

Enzymes: cystathionine c-synthase (EC 4.2.99.9; Swiss Prot entry

P55217); cystathionine b-lyase (EC 4.4.1.8; Swiss Prot entry P53780);

homoserine kinase (EC 2.7.1.39; Swiss Prot entry Q8L7R2); threonine

deaminase (EC 4.2.1.16; Swiss Prot entry Q9ZSS6); threonine synthase

(EC 4.2.99.2; Swiss Prot entry Q9S7B5); lactate dehydrogenase

(EC 1.1.1.27, Swiss Prot entry P13491).

Note: The mathematical model described here has been submitted to

the Online Cellular Systems Modelling Database and can be accessed

at http://jjj.biochem.sun.ac.za/database/curien/index.html free of

charge.

(Received 2 September 2003, accepted 23 September 2003)

Eur. J. Biochem. 270, 4615–4627 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03851.x