Acid-base Catalysis in the Mechanism of Thioredoxin Reductase from Drosophila melanogaster.

Abstract

Thioredoxin reductase (TrxR) catalyses the reduction of thioredoxin (Trx) by NADPH. Like other members of the pyridine nucleotide-disulfide family, TrxR is a homodimer. The catalytically active unit in the enzyme from Drosophila melanogaster (DmTrxR) consists of three redox centers: FAD and an N-terminal Cys-57/Cys-62 redox-active disulfide from one monomer, and a Cys-489’/Cys-490’ C-terminal redox-active disulfide from the second monomer. Because dipteran insects such as D. melanogaster lack glutathione reductase, glutathione disulfide must be reduced by Trx, making DmTrxR particularly important in this organism. DmTrxR is used as a model for the enzyme from a malaria vector, Anopheles gambiae. Based on the structures and mechanisms of other family members, a dyad of His-464’ and Glu-469’ acts as the acid-base catalyst of the dithiol-disulfide interchange reactions required in the catalysis of DmTrxR. The functions of His-464’ and Glu-469’ in the catalytic mechanism of DmTrxR were investigated. His-464’ was shown to be critical to catalysis by DmTrxR; thus, H464’Q retains only 2% of the wild-type activity. The pH dependence of Vmax for wild-type DmTrxR has apparent pKa values of 6.4 and 9.3, whereas H464’Q DmTrxR has an observable pKa only at 6.4, indicating that the pKa at pH 9.3 is contributed by His-464’. The macroscopic pKa at pH 6.4 has been assigned to Cys-57 and Cys-490’; the thiolate of Cys-57 is the nucleophile in the internal dithiol-disulfide interchange reaction and the thiolate of Cys-490’ is the nucleophile in the reduction of Trx. The rates of both the reductive and oxidative half reactions are markedly smaller in H464’Q DmTrxR than those of wild-type enzyme, indicating that His-464’ is involved in both half reactions. The pH dependence of the steady-state kinetics shows that the basicity of His-464’ decreases in the glutamate variants, as predicted. The reductive half-reactions of two glutamate variants are slower than those of wild-type enzyme. Malaria causes serious public health problems in the world. It is hoped that differences among TrxRs from human, Plasmodium falciparum (the causative agent) and Diptera (the vector) will be useful for developing differential inhibitors, useful as prophylactics.Ph.D.ToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/58421/1/huanghh_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/58421/2/huanghh_2.pd