METHIONINE BIOSYNTHESIS IN HIGHER-PLANTS .2. PURIFICATION AND CHARACTERIZATION OF CYSTATHIONINE BETA-LYASE FROM SPINACH-CHLOROPLASTS

Cystathionine beta-lyase, the second enzyme of the transsulfuration pa thway leading to homocysteine synthesis was purified over 16,000-fold from spinach (Spinacia oleracea L.) leaf chloroplasts (soluble fractio n). Enzyme activity was followed along the purification scheme by eith er a colorimetric method for the determination of cysteine or by fluor escence detection of the bimane derivative of L-homocysteine after rev erse-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 en zyme catalyzes alpha,beta-cleavage of the thioether L-cystathionine an d the dithioacetal L-djenkolate with apparent K-m values of 0.15 and 0 .34 mM, respectively, and apparent V-m values corresponding to a speci fic activity of 13 Units mg(-1). However, no activity was detected tow ard the disulfide L-cystine. With either L-cystathionine and L-djenkol ate as substrate, maximal activity was obtained between pH 8.3 and pH 9.0. Besides the chloroplastic enzyme form, anion exchange chromatogra phy 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 th e chloroplastic isoform. Kinetics of inactivation of cystathionine bet a-lyase by the L-alpha-(2-aminoethoxyvinyl) glycine (AVG), an analogue of L-cystathionine, are consistent with the existence of an intermedi ate reversible enzyme inhibitor complex (apparent inhibition constant K-i(app) of 110 mu M) preceding the irreversible formation of a final inactivated state of the enzyme (k(d) = 4.8 X 10(-3) s(-1)). Pyridoxal 5'-phosphate free in solution binds AVG with an apparent dissociation constant K-app in the order of 350 mu M. The comparison between the K -app (free pyridoxal 5'-phosphate) and K-i(app) (enzyme inactivation) values indicate that the prosthetic group of spinach chloroplast cysta thionine beta-lyase is freely accessible to the inhibitor compound AVG . (C) 1995 Academic Press, Inc.