Protein-protein interaction as a powering source of oxidoreductive reactivity.
Thiol-disulfide exchange reactions between thiol-disulfide oxidoreductases (e.g. thioredoxin or Trx) and client proteins can obtain a rate several orders faster than those between chemical reagents (e.g. dithiothreitol) and client proteins. The active sites of these oxidoreductases are characterized by a CXXC motif. The XX dipeptide of Trx is GP. By altering the C-terminal X to A, K and D, it is shown that the P --> K mutation confers the largest effect on the redox potential, which it elevated by 28 mV, while the P --> D mutation displays the smallest variation. The change in pK(a) of the nucleophilic thiol also follows this trend. However, GK and GA react faster with thioredoxin reductase, exhibiting a rate rank of GK > GA > GP > GD, while the rates toward insulin and PDI follow the order GP > GA > GK > GD. The rate change spans two to three orders of magnitude. This work demonstrates that redox reactivity does not correlate simply with pK(a) and redox potential, but instead supports the important role of interaction between proteins in determining the fast reactivity and rate order of Trx. A reaction mechanism involving the transient formation of a Trx-protein binding complex is proposed for the oxidoreduction of protein thiols-disulfides. Furthermore, studies on insulin reduction show that Trx acts as an enzyme rather than a redox couple. These results provide explanations for the observed variations of the CXXC motif in PDI-like proteins as well as the conservation of the CXXC motif in Trx.
Lin
Tiao-Yin
TY
Department of Biological Science and Technology, National Chiao Tung University, 75 Poai St., Hsinchu, Taiwan, Republic of China. tylin@cc.nctu.edu.tw
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004734:Energy Metabolism / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D050505:Mutant Proteins; D011506:Proteins; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D011490:Protein Disulfide Reductase (Glutathione); D019704:Protein Disulfide-Isomerases