Purification and characterization of O-acetylserine sulfhydrylase isoenzymes from Datura innoxia.
Three isoenzyme forms (designated A, B, and C) of O-acetylserine sulfhydrylase were purified from Datura innoxia suspension cultures. Isoenzyme A is the most abundant form, comprising 45-60% of the total activity. Isoenzymes C and B comprise 35-40% and 10-20% of the activity, respectively. The specific activities of the purified isoenzymes are similar (870-893 mumol of cysteine/min/mg of protein). Molecular masses for isoenzymes A, B, and C, estimated by analytical size exclusion high performance liquid chromatography, are 63, 86, and 63 kDa, respectively. Isoenzymes A and B are homodimers; isoenzyme C is a heterodimer. Spectral analysis indicates that these isoenzymes possess a pyridoxal 5'-phosphate cofactor that binds the O-acetylserine substrate. Binding is reversible by addition of the sulfide substrate. The O-acetylserine sulfhydrylase isoenzymes are active over a broad temperature range, with maximum activity between 42 and 58 degrees C. They are active only between pH 7 and 8, with optimal activity at pH 7.6. Kinetic analysis indicates these enzymes are allosterically regulated and exhibit positive cooperativity with respect to both substrates. They are inhibited by sulfide concentrations above 200 microM. The kinetic analysis together with the physical and spectrophotometric characteristics indicate that the O-acetylserine sulfhydrylase enzymes have two active sites.
Kuske
C R
CR
Genomics and Structural Biology, Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545.
Ticknor
L O
LO
Guzmán
E
E
Gurley
L R
LR
Valdez
J G
JG
Thompson
M E
ME
Jackson
P J
PJ
D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003547:Cysteine Synthase / Q000302:isolation & purification* / Q000378:metabolism; D013286:Datura stramonium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010946:Plants, Medicinal; D010947:Plants, Toxic; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013440:Sulfides / Q000378:metabolism; D013696:Temperature
D007527:Isoenzymes; D013440:Sulfides; D012694:Serine; D003547:Cysteine Synthase; C043943:O-acetylserine; C033479:sodium sulfide