PURIFICATION AND KINETIC-PROPERTIES OF SERINE ACETYLTRANSFERASE FREE OF O-ACETYLSERINE(THIOL)LYASE FROM SPINACH-CHLOROPLASTS

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Sp inacia oleracea) leaf chloroplasts. The purification procedure consist ed of ammonium sulfate precipitation, anion-exchange chromatography (T risacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, a nd gel filtration (Superdex 200). The purified enzyme exhibited a spec ific activity higher than 200 units mg(-1) and a subunit molecular mas s of about 33 kD upon polyacrylamide get electrophoresis in the presen ce of sodium dodecyl sulfate. Moreover, the purified serine acetyltran sferase appeared to be essentially free of O-acetylserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A st eady-state kinetic analysis indicated that the mechanism of the enzyme -catalyzed reaction involves a double displacement. The apparent K, fo r the two substrates, L-serine and acetyl-coenzyme A, were 2.29 +/- 0. 43 and 0.35 +/- 0.02 mM, respectively. The rate of L-cysteine synthesi s in vitro was measured in a coupled enzyme assay using extensively pu rified O-acetylserine(thiol)lyase and serine acetyltransferase. This r ate was maximum when the assay contained approximately a 400-fold exce ss of O-acetylserine(thiol)lyase over serine acetyltransferase. Measur ements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former e nzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase acti vity/serine acetyltransferase activity] measured in the stromal protei n extract was 345. This strongly suggested that all the O-acetylserine (thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplas t.