Methionine Biosynthesis in Higher Plants .II. Purification and Characterization of Cystathionine β-Lyase from Spinach Chloroplasts

Abstract

Cystathionine β-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 β-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 α,β-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-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 β-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 β-lyase by the L-α-(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 μM) preceding the irreversible formation of a final inactivated state of the enzyme (kd = 4.8 × 10−3 s−1). Pyridoxal 5′-phosphate free in solution binds AVG with an apparent dissociation constant Kapp in the order of 350 μM. The comparison between the Kapp (free pyridoxal 5′-phosphate) and Kappi (enzyme inactivation) values indicate that the prosthetic group of spinach chloroplast cystathionine β-lyase is freely accessible to the inhibitor compound AVG.