O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Further purification and characterization as a pyridoxal enzyme.
O-Acetylserine-O-acetylhomoserine sulfhydrylase [EC class 4.2.99], catalyzing the sulfhydrylation of both O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) (O-acetyl-L-serine(O-acetyl-L-homoserine) + H2S leads to L-cysteine (L-homocysteine) + acetate), was extracted and purified from bakers' yeast by an improved method. The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis both in the absence and presence of sodium dodecylsulfate and by ultracentrifugal analysis. The apo-enzyme was protected by pyridoxal phosphate (PALP) from inactivation by heat, urea, and trypsin [EC 3.4.21.4], suggesting that the binding of PALP to the apo-enzyme rendered the conformation of the protein more stable. The holo-enzyme showed absorption peaks at 420 and 330 nm due to bound PALP, in addition to a peak at 280 nm. Upon reduction with borohydride, the 420-nm peak disappeared and an increase in the 330-nm peak occurred concomitant with loss of the catalytic activity. Lysine appeared to be the pyridoxal binding site, based on identification of pyridoxyl-lysine in the hydrolyzate of the holo-enzyme. It was shown by both spectral and chemical determinations that 4 moles of PALP could bind to 200,000 g of apo-protein. The apo-enzyme showed a lower association constant with PALP than some other enzymes. Pyridoxal inhibited the activity competitively with respect to PALP. Based on these findings, it appears that the reaction mechanism of this enzyme is similar to those of other pyridoxal enzymes.
D003547:Cysteine Synthase / Q000302:isolation & purification / Q000378:metabolism; D004355:Drug Stability; D006714:Homoserine / Q000031:analogs & derivatives; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D011730:Pyridoxal; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014357:Trypsin; D014508:Urea
D009097:Multienzyme Complexes; D011730:Pyridoxal; D006714:Homoserine; D014508:Urea; D003547:Cysteine Synthase; D014357:Trypsin; D008190:Lyases