Yeast thioltransferase--the active site cysteines display differential reactivity.
Thioltransferase, catalyzing thiol-disulfide interchange between reduced glutathione and disulfides, was purified to homogeneity from Saccharomyces cerevisiae. The purification procedure included ammonium sulfate precipitation, Sephadex G-50 gel filtration, CM-Sepharose ion exchange chromatography, and C18 reverse phase high pressure liquid chromatography. Two thioltransferase activity peaks were resolved by CM-Sepharose chromatography. The protein from the major peak had a molecular weight of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis while the minor peak protein migrated slightly faster in this gel system. Both proteins showed similar amino acid compositions and identical N-termini. The major peak of thioltransferase was extensively characterized. Plots of thioltransferase activity as a function of S-sulfocysteine or hydroxyethyl disulfide concentration did not show normal Michaelis-Menten kinetics. The enzyme activity had a pH optimum of 9.1. The protein has 106 amino acid residues with two cysteines and no arginine. The active site amino acid sequence of the enzyme was identified as Cys26-Pro-Tyr-Cys29, which is similar to that of mammalian thioltransferase and Escherichia coli glutaredoxin. The two cysteines at the active site displayed different reactivities to iodoacetamide. Cys26 was alkylated by iodoacetamide at pH 3.5 while Cys29 was alkylated at pH 8.0. The enzyme was completely inactivated when the Cys26 was carboxymethylated. A plot of incorporation of iodoacetamide into Cys29 at different pHs was similar to the pH dependence of the enzyme activity. The result suggested that Cys26 could readily initiate nucleophilic attack on disulfide substrates at physiological pH.
Gan
Z R
ZR
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Sardana
M K
MK
Jacobs
J W
JW
Polokoff
M A
MA
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011490:Protein Disulfide Reductase (Glutathione); D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000596:Amino Acids; D054477:Glutaredoxins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D003545:Cysteine