title: The pKa value and accessibility of cysteine residues are key determinants for protein substrate discrimination by glutaredoxin.
abstract: The enzyme glutaredoxin catalyzes glutathione exchange, but little is known about its interaction with protein substrates. Very different proteins are substrates in vitro, and the enzyme seems to have low requirements for specific protein interactions. Here we present a systematic investigation of the interaction between human glutaredoxin 1 and glutathionylated variants of a single model protein. Thus, single cysteine variants of acyl-coenzyme A binding protein were produced creating a set of substrates in the same protein background. The rate constants for deglutathionylation differ by more than 2 orders of magnitude between the best (k1 = 1.75 × 10(5) M(-1) s(-1)) and the worst substrate (k1 = 4 × 10(2) M(-1) s(-1)). The pKa values of the substrate cysteine residues were determined by NMR spectroscopy and found to vary from 8.2 to 9.9. Rates of glutaredoxin 1-catalyzed deglutathionylation were assessed with respect to substrate cysteine pKa values, cysteine residue accessibility, local stability, and backbone dynamics. Good substrates are characterized by a combination of high accessibility of the glutathionylated site and low pKa of the cysteine residue.
authors.affiliation: Department of Biology, University of Copenhagen , Ole Maaleøs Vej 5, 2200 Copenhagen N, Denmark.
authors.forename: Kristine Steen
authors.initials: KS
authors.lastname: Jensen
authors.forename: Jeppe T
authors.initials: JT
authors.lastname: Pedersen
authors.forename: Jakob R
authors.initials: JR
authors.lastname: Winther
authors.forename: Kaare
authors.initials: K
authors.lastname: Teilum
mesh_terms: D055162:Biocatalysis; D003545:Cysteine / Q000378:metabolism*; D054477:Glutaredoxins / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular
chemical_list: D054477:Glutaredoxins; D003545:Cysteine