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 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 K-m values of 0.15 and 0.34 mM, respectively, and apparent V-m values corresponding to a specific activity of 13 Units mg(-1). However, no activity was detected toward the disulfide L-cystine. 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 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 cystathionine beta-lyase is freely accessible to the inhibitor compound AVG. (C) 1995 Academic Press, Inc.