Plasmid ptyb3 and restriction enzymes

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)).

NADPH was purchased from
AppliChem (Darmstadt,
Germany). DEAE resin was obtained from Sigma-Aldrich (St. Louis, MO).
Phenyl sepharose resin was from Pharmacia-Amersham Biosciences (Uppsala,
Sweden). Microcon Ultracel YM-50 ultrafiltration devices by Millipore
(Billerica, MA) were used for concentrating enzyme samples. 2-Chlorotritylchloride
resin 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). CLEAR-OX resin was from
Peptides International (Louisville, KY). Primers for DmTR mutants
were from IDT (Coralville, IA), and plasmid pTYB3 and restriction
enzymes were from New England Biolabs (Ipswich, MA). The DmTR-SG construct
was synthesized by Retrogen, Inc. (San Diego, CA). 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 (Fair Lawn, NJ) or Acros Organics (Morris Plains,
NJ).