Thioredoxins catalyze thiol/disulfide redox reactions for a wide range of small molecules and protein substrates. In addition to biosynthetic roles in ribonucleotide reduction and reduction of inorganic sulfate, thioredoxins are involved in a wide range of regulatory functions in mammalian systems including activation of transcription factors NF-kB and AP-1 and the glucocorticoid receptor. The human protein disulfide isomerase utilizes two thioredoxin-like domains in its crucial role in maturation of secreted proteins. However, despite a striking similarity in the conformation of the active sites of the thioredoxins and the protein disulfide isomerases, the redox equilibria of the two proteins differs by 104 yielding physiological roles as protein reductants and protein oxidants/disulfide exchange catalysts, respectively. Previous work of this lab has indicated that the kinetic control occurs in the breakdown of the mixed disulfide intermediate and that the buried acid sidechain common to these proteins functions in the formation/breakdown of this intermediate. Stopped flow fluorescence and absorbance spectroscopy will be used to determine the rate constants of the individual reaction steps for a series of active site mutations in the genetic backgrounds of both the E. coli and human thioredoxins as well as for the human protein disulfide isomerase thioredoxin-like domains. These data will be complemented by nuclear magnetic resonance measurements of reaction intermediate concentrations and structural characterization of the variant enzymes. The proposed experiments will provide further insight into mechanistic role of several evolutionarily conserved residues in the active site. Detailed understanding of the mechanistic regulation of this family of proteins may serve not only for potential therapeutic applications but also in the development of catalysts for in vitro folding of heterologously expressed proteins of medical and industrial significance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057266-03
Application #
6386847
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
1999-09-01
Project End
2004-08-31
Budget Start
2001-09-01
Budget End
2004-08-31
Support Year
3
Fiscal Year
2001
Total Cost
$238,230
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
110521739
City
Menands
State
NY
Country
United States
Zip Code
12204