Identification of residues of spinach thioredoxin f that influence interactions with target enzymes.
The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood. To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes. Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77. Kinetics of activation of oxidized recombinant sorghum leaf NADP-dependent malate dehydrogenase and oxidized spinach chloroplastic fructose-1,6-bisphosphatase by wild-type Trx f, wild-type Trx m, and Trx f mutants were compared. All of the mutants are less efficient than wild-type Trx f in the activation of fructose-1,6-bisphosphatase and are altered in both S0.5 and Vmax. In contrast to literature reports, the activation of NADP-dependent malate dehydrogenase does not display rate saturation kinetics with respect to the concentration of Trx f, thereby signifying very weak interactions between the two proteins. The mutants of Trx f likewise interact only weakly with NADP-dependent malate dehydrogenase, but the apparent second-order rate constants for activation are increased compared to that with wild-type Trx f. Thus, Lys-58, Asn-74, Gln-75, and Asn-77 of Trx f contribute to its interaction with target enzymes and influence target protein selectivity.
Geck
M K
MK
University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37831, USA.
Larimer
F W
FW
Hartman
F C
FC
D000595:Amino Acid Sequence; D054479:Chloroplast Thioredoxins; D004789:Enzyme Activation; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology / Q000378:metabolism*; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D054479:Chloroplast Thioredoxins; D010940:Plant Proteins; D013879:Thioredoxins; D008291:Malate Dehydrogenase; D006597:Fructose-Bisphosphatase