title: Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, HvTrxh1 and HvTrxh2.
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 approximately 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 approximately 70% higher reactivity in HvTrxh1, suggesting significant functional difference between the isozymes.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Kenji
authors.initials: K
authors.lastname: Maeda
authors.forename: Per
authors.initials: P
authors.lastname: Hägglund
authors.forename: Olof
authors.initials: O
authors.lastname: Björnberg
authors.forename: Jakob R
authors.initials: JR
authors.lastname: Winther
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013816:Thermodynamics; D054480:Thioredoxin h / Q000737:chemistry / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D007328:Insulin; D007527:Isoenzymes; D010940:Plant Proteins; D013438:Sulfhydryl Compounds; D054480:Thioredoxin h; D003545:Cysteine