TY - JOUR

T1 - The pKa value and accessibility of cysteine residues are key determinants for protein substrate discrimination by glutaredoxin

AU - Jensen, Kristine Steen

AU - Pedersen, Jeppe Trudslev

AU - Winther, Jakob R.

AU - Teilum, Kaare

PY - 2014

Y1 - 2014

N2 - 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 × 105 M–1 s–1) and the worst substrate (k1 = 4 × 102 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.

AB - 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 × 105 M–1 s–1) and the worst substrate (k1 = 4 × 102 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.

U2 - 10.1021/bi4016633

DO - 10.1021/bi4016633

M3 - Journal article

C2 - 24673564

VL - 53

SP - 2533

EP - 2540

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 15

ER -