Methionine Biosynthesis in Higher-Plants .1. Purification and Characterization of Cystathionine γ-Synthase from Spinach Chloroplasts

Cystathionine γ-synthase, the first enzyme specific for the methionine biosynthetic pathway, was purified to apparent homogeneity from spinach leaf chloroplasts. A nonradioactive assay based on O-phthaldialdehyde derivatization of L-cystathionine and fluorescence detection was developed to determine the cystathionine γ-synthase activity. A unique cystathionine γ-synthase activity was located in the stromal fraction of chloroplasts while cystathionine β-lyase, the second enzyme of the transsulfuration pathway, was associated with both the chloroplastic and cytosolic compartments (see companion manuscript). The purified enzyme exhibited a specific activity of 13 U mg−1. As estimated by gel filtration and polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by activity staining, the native enzyme had an apparent Mr of 215,000. On the basis of sodium dodecyl sulfate-PAGE, purified cystathionine γ-synthase migrated as two molecular species of Mr 53,000 and 50,000 that are identical in their N-termini. The absorption spectrum obtained at pH 7.5 exhibited a peak at 425 nm due to pyridoxal 5′-phosphate (PLP). The purified enzyme catalyzed the formation of L-cystathionine or L-homocysteine depending on the sulfur-containing substrate, L-cysteine or sulfide. Maximal cystathionine γ-synthase activity was found at pH 7.4. The apparent Km values for O-phospho-L-homoserine (the unique homoserine ester synthesized in the chloroplast), L-cysteine, and sulfide were 1.4, 0.18, and 0.6 mM, respectively. Inactivation of cystathionine γ-synthase by DL-propargylglycine (PAG) showed pseudo-first-order kinetics and data were consistent with the existence of an intermediate reversible enzyme-inhibitor complex (Kappi = 140 μM) preceding the formation of a final enzyme-inhibitor complex (kd = 24 × 10−3 s−1). The irreversibility of the inhibition and the partial restoration of the activity by pyridoxal-phosphate suggest that PAG interacts with the PLP prosthetic group of the enzyme. Kinetic and equilibrium binding studies showed that PAG binding to PLP was considerably enhanced in the enzyme binding pocket compared to that with PLP free in solution.