Methionine biosynthesis in higher plants. II. Purification and characterization of cystathionine beta-lyase from spinach chloroplasts.
Cystathionine beta-lyase, the second enzyme of the transsulfuration pathway leading to homocysteine synthesis was purified over 16,000-fold from spinach (Spinacia oleracea L.) leaf chloroplasts (soluble fraction). Enzyme activity was followed along the purification scheme by either a colorimetric method for the determination of cysteine or by fluorescence detection of the bimane derivative of L-homocysteine after reverse-phase HPLC. Cystathionine beta-lyase has a molecular mass of 170,000 +/- 5000 Da and consists of four identical subunits of 44,000 Da. The enzyme exhibits an absorption spectrum in the visible range with a maximum at 418 nm due to pyridoxal 5'-phosphate. The chloroplastic enzyme catalyzes alpha,beta-cleavage of the thioether L-cystathionine and the dithioacetal L-djenkolate with apparent Km values of 0.15 and 0.34 mM, respectively, and apparent Vm values corresponding to a specific activity of 13 Units mg-1. However, no activity was detected toward the disulfide L-cysteine. With either L-cystathionine and L-djenkolate as substrate, maximal activity was obtained between pH 8.3 and pH 9.0. Besides the chloroplastic enzyme form, anion exchange chromatography of a total spinach leaf extract allowed the detection of a second pool of cystathionine beta-lyase activity that is associated with the cytosolic compartment and eluted at a lower salt concentration than the chloroplastic isoform. Kinetics of inactivation of cystathionine beta-lyase by the L-alpha-(2-aminoethoxyvinyl) glycine (AVG), an analogue of L-cystathionine, are consistent with the existence of an intermediate reversible enzyme inhibitor complex (apparent inhibition constant Kappi of 110 microM) preceding the irreversible formation of a final inactivated state of the enzyme (kd = 4.8 x 10(-3) s-1). Pyridoxal 5'-phosphate free in solution binds AVG with an apparent dissociation constant Kapp in the order of 350 microM. The comparison between the Kapp (free pyridoxal 5'-phosphate) and Kappi (enzyme inactivation) values indicate that the prosthetic group of spinach chloroplast cystathionine beta-lyase is freely accessible to the inhibitor compound AVG.
Droux
M
M
Unité Mixte CNRS/Rhône-Poulenc Agrochimie, Centre de la Recherche de la Dargoire, Lyon, France.
Ravanel
S
S
Douce
R
R
D000613:Aminobutyrates / Q000494:pharmacology; D002451:Cell Compartmentation; D002736:Chloroplasts / Q000201:enzymology*; D003540:Cystathionine / Q000378:metabolism*; D006710:Homocysteine / Q000032:analysis / Q000096:biosynthesis*; D007700:Kinetics; D008190:Lyases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000096:biosynthesis*; D018515:Plant Leaves / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011773:Pyruvates / Q000032:analysis; D019289:Pyruvic Acid; D013053:Spectrophotometry; D018724:Spinacia oleracea / Q000201:enzymology
D000613:Aminobutyrates; D011773:Pyruvates; D006710:Homocysteine; D003540:Cystathionine; C012175:2-amino-4-(2-aminoethoxy)butanoic acid; D011732:Pyridoxal Phosphate; D019289:Pyruvic Acid; D008715:Methionine; D008190:Lyases; C019965:cystathionine beta-lyase