Biochemical characterization of dithiol glutaredoxin 8 from Saccharomyces cerevisiae: the catalytic redox mechanism redux.
Two dithiol glutaredoxins (Grxs), Grx1 and Grx2, from yeast have been characterized to date. A third putative dithiol glutaredoxin-encoding gene (GRX8) has been identified in silico. Here we show that deletion of GRX8 does not result in a reduced growth rate under oxidative stress conditions, nor does it enhance the defects of Deltagrx1 and Deltagrx2 single or double mutants. We furthermore compare the enzymatic properties of recombinant ScGrx8 with the monothiol glutaredoxin ScGrx7. Molecular models of ScGrx8 suggest that the protein has a canonical Grx fold, a significantly altered substrate binding site, and a Trp14-type cysteine motif at the catalytic center. ScGrx8 did not bind heavy metal ions and was exclusively monomeric. Apparent k(cat) values for ScGrx8 in the standard enzymatic assay were about 3 orders of magnitude less than for ScGrx7, whereas apparent K(m) values were comparable. Mass spectrometric analyses support a ping-pong mechanism for ScGrx7 and ScGrx8 with a glutathionylated protein as an intermediate. Reduction kinetics of ScGrx8 disulfide, glutathionylated ScGrx8(C28S), and glutathionylated ScGrx7 revealed significant differences between the proteins. Surprisingly, mutation of the more C-terminal cysteine residue in the CPDC motif of ScGrx8 also abolished the slight enzymatic activity, and thus the standard catalytic mechanism for glutathionylated substrates does not apply to the enzyme. In summary, ScGrx8 has several novel structural and mechanistic features expanding the subclasses of glutaredoxins. A refined catalytic model for monothiol and dithiol glutaredoxins is presented explaining the diversity of enzymatic activities in vitro and pointing to different functions in vivo.
Eckers
Elisabeth
E
Butenandt Institute for Physiological Chemistry, Ludwig-Maximilians University, D-81377 Munich, Germany.
Bien
Melanie
M
Stroobant
Vincent
V
Herrmann
Johannes M
JM
Deponte
Marcel
M
D000595:Amino Acid Sequence; D055162:Biocatalysis; D001681:Biological Assay; D020134:Catalytic Domain; D003001:Cloning, Molecular; D054477:Glutaredoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism
D054477:Glutaredoxins; D007328:Insulin; D013438:Sulfhydryl Compounds; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione