SPINACH CHLOROPLAST O-ACETYLSERINE (THIOL)-LYASE EXHIBITS 2 CATALYTICALLY NONEQUIVALENT PYRIDOXAL-5'-PHOSPHATE-CONTAINING ACTIVE-SITES

A synthetic gene encoding the mature spinach-chloroplast O-acetylserin e (thiol)-lyase was constructed and expressed in an Escherichia coli s train carrying the T7 RNA polymerase system. The pure recombinant prot ein was obtained at high yield (6 mg/l cell culture) using a new purif ication procedure that includes affinity chromatography on Green A aga rose. Its specific activity was of the order of 1000 U/mg, and its phy sical properties were similar to those previously reported for the nat ural enzyme isolated from spinach chloroplasts. In particular the reco mbinant enzyme, as for the natural enzyme, behaved as a homodimer comp osed of two identical subunits each of M(r) 35000. From steady-state k inetic studies using sulfide or 5-thio(2-nitrobenzoate) (Nbs) as alter native nucleophilic co-substrates, the enzyme exhibited positive kinet ic co-operativity with respect to O-acetylserine [Ser(Ac)] in the pres ence of sulfide and a negative kinetic co-operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by abso rbance and fluorescence measurements to obtain insight into the role o f pyridoxal 5'-phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme pro voked the disappearance of the 409-nm absorbance band of the pyridoxal 5'-phosphate Schiff base and the appearance of two new absorbance ban ds, the one located between 320 nm and 360 nm and the other centered a t 470 nm. Also, the fluorescence emission of the pyridoxal 5'-phosphat e Schiff base was quenched upon addition of Ser(Ac) to the enzyme. The se changes were most presumably due to the formation of a Schiff base intermediate between alpha-aminoncrylate and the pyridoxal 5'-phosphat e cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbanc e and fluorescence equilibrium data disclosed a co-operative behavior in Ser(Ac) binding, in agreement with the steady-state kinetic results . Fluorescence quenching experiments with acrylamide and iodide reveal ed that the indole ring of Trp176 was largely exposed and located with in the pyridoxal 5'-phosphate active site. These results are consisten t with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit-subunit interactions, the en zyme exhibits two non-equivalent pyridoxal-5'-phosphate-containing act ive sites.