Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, HvTrxh1 and HvTrxh2

TY - JOUR

T1 - Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, HvTrxh1 and HvTrxh2

AU - Maeda, Kenji

AU - Hägglund, Per

AU - Björnberg, Olof

AU - Winther, Jakob R

AU - Svensson, Birte

N1 - Keywords: Thioredoxin; Dithiol/disulfide exchange; Tryptophan fluorescence; Redox potential; Thiol pKa

PY - 2010/8

Y1 - 2010/8

N2 - Barley thioredoxin h isozymes 1 (HvTrxh1) and barley thioredoxin h isozymes 2 (HvTrxh2) show distinct spatiotemporal distribution in germinating seeds. Using a novel approach involving measurement of bidirectional electron transfer rates between Escherichia coli thioredoxin, which exhibits redox-dependent fluorescence, and the barley isozymes, reaction kinetics and thermodynamic properties were readily determined. The reaction constants were ∼60% higher for HvTrxh1 than HvTrxh2, while their redox potentials were very similar. The primary nucleophile, CysN, of the active site Trp-CysN-Gly-Pro-CysC motif has an apparent pKa of 7.6 in both isozymes, as found by iodoacetamide titration, but showed ∼70% higher reactivity in HvTrxh1, suggesting significant functional difference between the isozymes.

AB - Barley thioredoxin h isozymes 1 (HvTrxh1) and barley thioredoxin h isozymes 2 (HvTrxh2) show distinct spatiotemporal distribution in germinating seeds. Using a novel approach involving measurement of bidirectional electron transfer rates between Escherichia coli thioredoxin, which exhibits redox-dependent fluorescence, and the barley isozymes, reaction kinetics and thermodynamic properties were readily determined. The reaction constants were ∼60% higher for HvTrxh1 than HvTrxh2, while their redox potentials were very similar. The primary nucleophile, CysN, of the active site Trp-CysN-Gly-Pro-CysC motif has an apparent pKa of 7.6 in both isozymes, as found by iodoacetamide titration, but showed ∼70% higher reactivity in HvTrxh1, suggesting significant functional difference between the isozymes.

U2 - 10.1016/j.febslet.2010.06.028

DO - 10.1016/j.febslet.2010.06.028

M3 - Journal article

C2 - 20594550

SN - 0014-5793

VL - 584

SP - 3376

EP - 3380

JO - FEBS Letters

JF - FEBS Letters

IS - 15

ER -