Reference "Methionine biosynthesis in higher plants. I. Purification and characterization of cystathionine gamma-synthase from spinach chloroplasts"

Reference ID

54993

Title

Methionine biosynthesis in higher plants. I. Purification and characterization of cystathionine gamma-synthase from spinach chloroplasts

Source

Arch Biochem Biophys, 1995, vol. 316, pp. 572-584

Authors (3)

Ravanel-S	Droux-M
Douce-R

Abstract

Cystathionine gamma-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 gamma-synthase activity. A unique cystathionine gamma-synthase
activity was located in the stromal fraction of chloroplasts while cystathionine
beta-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 M(r) of 215,000. On the basis of sodium dodecyl sulfate-PAGE, purified
cystathionine gamma-synthase migrated as two molecular species of M(r) 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 gamma-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 gamma-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 microM) preceding the formation of a final enzyme-inhibitor
complex (kd = 24 x 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.