Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase.
Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.
Sun
Qi-An
QA
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Su
Dan
D
Novoselov
Sergey V
SV
Carlson
Bradley A
BA
Hatfield
Dolph L
DL
Gladyshev
Vadim N
VN
D000818:Animals; D054477:Glutaredoxins; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism; D014018:Tissue Distribution
D054477:Glutaredoxins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D013879:Thioredoxins; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; C466433:thioredoxin glutathione reductase; D012643:Selenium