Structural and biochemical studies reveal differences in the catalytic mechanisms of mammalian and Drosophila melanogaster thioredoxin reductases.

摘要: Thioredoxin reductase (TR) from Drosophila melanogaster (DmTR) is a member of the glutathione (GR) family pyridine nucleotide disulfide oxidoreductases and catalyzes reduction redox-active bond thioredoxin. DmTR notable for having high catalytic activity without presence selenocysteine (Sec) residue (which essential mammalian thioredoxin reductases). We report here X-ray crystal structure at 2.4 A resolution (Rwork = 19.8%, Rfree 24.7%) in which enzyme was truncated to remove C-terminal tripeptide sequence Cys-Cys-Ser. also demonstrate that tetrapeptides equivalent oxidized active sites both mouse mitochondrial TR (mTR3) are substrates forms enzymes. This enzyme/peptide substrate system examines kinetics ring-opening step occurs during enzymatic cycle TR. The 300-500-fold slower when Sec replaced with Cys mTR3 using this system. Conversely, DmTR, rate ring opening only moderately increased (5-36-fold). Structures these were oriented site enzymes bound GR as template. has more open tetrapeptide binding pocket than accommodates peptide Ser-Cys-Cys-Ser(ox) cis conformation allows protonation leaving-group by His464', helps explain why can function need Sec. In contrast, shows narrower pocket. One possible result interface Gly-Cys-Sec-Gly may adopt trans better fit. places farther away protonating histidine residue, but lower pKa comparison eliminates be protonated.