NADPH was purchased from
AppliChem (Darmstadt,
Germany). Dithionitrobenzoic acid (DTNB), sodium selenite, and DEAE
resin were all obtained from Sigma-Aldrich (St. Louis, MO). Phenyl
Sepharose resin was from Pharmacia-Amersham Biosciences (Uppsala,
Sweden). Microcon Ultracel YM-50 ultrafiltration devices from Millipore
(Billerica, MA) were used for concentrating enzyme samples. Resin
for peptide synthesis (2-chlorotritylchloride) was from Novabiochem
(San Diego, CA). Fmoc amino acids were from Synbiosci Corp. (Livermore,
CA), except for Fmoc-homocysteine, which was from Bachem (King of
Prussia, PA). Primers for mTR3 mutants were from IDT (Coralville,
IA). Plasmid pTYB3 and restriction enzymes were from New England Biolabs
(Ipswich, MA). The production and purification of the recombinant
and semisynthetic enzymes used in this study have been previously
reported.6 (link),19 (link),24 (link),25 (link),30 (link) The selenium content
of the wild-type (WT) semisynthetic enzyme is 91% as reported in ref (19 (link)). Enzyme kinetic assays
were performed on a Cary50 UV–vis spectrophotometer (Walnut
Creek, CA), and all enzymatic assays were conducted at room temperature
unless otherwise noted. All other chemicals were from Fisher Scientific
or Acros Organics (Morris Plains, NJ). Aryl disulfides were prepared
by Watson Lees and others as described in refs (31 (link)−35 (link)).
AppliChem (Darmstadt,
Germany). Dithionitrobenzoic acid (DTNB), sodium selenite, and DEAE
resin were all obtained from Sigma-Aldrich (St. Louis, MO). Phenyl
Sepharose resin was from Pharmacia-Amersham Biosciences (Uppsala,
Sweden). Microcon Ultracel YM-50 ultrafiltration devices from Millipore
(Billerica, MA) were used for concentrating enzyme samples. Resin
for peptide synthesis (2-chlorotritylchloride) was from Novabiochem
(San Diego, CA). Fmoc amino acids were from Synbiosci Corp. (Livermore,
CA), except for Fmoc-homocysteine, which was from Bachem (King of
Prussia, PA). Primers for mTR3 mutants were from IDT (Coralville,
IA). Plasmid pTYB3 and restriction enzymes were from New England Biolabs
(Ipswich, MA). The production and purification of the recombinant
and semisynthetic enzymes used in this study have been previously
reported.6 (link),19 (link),24 (link),25 (link),30 (link) The selenium content
of the wild-type (WT) semisynthetic enzyme is 91% as reported in ref (19 (link)). Enzyme kinetic assays
were performed on a Cary50 UV–vis spectrophotometer (Walnut
Creek, CA), and all enzymatic assays were conducted at room temperature
unless otherwise noted. All other chemicals were from Fisher Scientific
or Acros Organics (Morris Plains, NJ). Aryl disulfides were prepared
by Watson Lees and others as described in refs (31 (link)−35 (link)).