Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, HvTrxh1 and HvTrxh2 |
| |
Authors: | Kenji Maeda,Per Hä gglund,Jakob R. Winther |
| |
Affiliation: | a Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark b Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark |
| |
Abstract: | 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. |
| |
Keywords: | Thioredoxin Dithiol/disulfide exchange Tryptophan fluorescence Redox potential Thiol pKa |
本文献已被 ScienceDirect 等数据库收录! |
|