METHIONINE BIOSYNTHESIS IN HIGHER-PLANTS .1. PURIFICATION AND CHARACTERIZATION OF CYSTATHIONINE GAMMA-SYNTHASE FROM SPINACH-CHLOROPLASTS

Cystathionine gamma-synthase, the first enzyme specific for the methio nine biosynthetic pathway, was purified to apparent homogeneity from s pinach leaf chloroplasts. A nonradioactive assay based on O-phthaldial dehyde derivatization of L-cystathionine and fluorescence detection wa s developed to determine the cystathionine gamma-synthase activity. A unique cystathionine gamma-synthase activity was located in the stroma l fraction of chloroplasts while cystathionine beta-lyase, the second enzyme of the transsulfuration pathway, was associated with both the c hloroplastic and cytosolic compartments (see companion manuscript). Th e purified enzyme exhibited a specific activity of 13 U mg(-1). As est imated by gel filtration and polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by activity staining, the nat ive enzyme had an apparent M(r) of 215,000. On the basis of sodium dod ecyl sulfate-PAGE, purified cystathionine gamma-synthase migrated as t wo molecular species of M(r) 53,000 and 50,000 that are identical in t heir N-termini. The absorption spectrum obtained at pH 7.5 exhibited a peak at 425 nm due to pyridoxal 5'-phosphate (PLP). The purified enzy me catalyzed the formation of L-cystathionine or L-homocysteine depend ing on the sulfur-containing substrate, L-cysteine or sulfide. Maximal cystathionine gamma-synthase activity was found at pH 7.4. The appare nt K-m values for O-phospho-L-homoserine (the unique homoserine ester synthesized in the chloroplast), L-cysteine, and sulfide were 1.4, 0.1 8, and 0.6 mM, respectively. Inactivation of cystathionine gamma-synth ase by DL-propargylglycine (FAG) showed pseudo-first-order kinetics an d data were consistent with the existence of an intermediate reversibl e enzyme-inhibitor complex (K-i(app) = 140 mu M) preceding the formati on of a final enzyme-inhibitor complex (k(d) = 24 X 10(-3) s(-1)). The irreversibility of the inhibition and the partial restoration of the activity by pyridoxal-phosphate suggest that FAG interacts with the PL P prosthetic group of the enzyme. Kinetic and equilibrium binding stud ies showed that FAG binding to PLP was considerably enhanced in the en zyme binding pocket compared to that with PLP free in solution. (C) 19 95 Academic Press, Inc.