Catalytic properties of selenophosphate synthetases: comparison of the selenocysteine-containing enzyme from Haemophilus influenzae with the corresponding cysteine-containing enzyme from Escherichia coli.
The selD gene from Haemophilus influenzae has been overexpressed in Escherichia coli. The expressed protein was purified to homogeneity in a four-step procedure and then carboxymethylated by reaction with chloroacetate. N-terminal sequencing by Edman degradation identified residue 16 as carboxymethyl selenocysteine, which corresponded to the essential cysteine residue in the glycine-rich sequence of the E. coli selenophosphate synthetase. It would be expected that an ionized selenol of a selenocysteine in place of a catalytically essential cysteine residue would result in an enzyme with increased catalytic activity. To test this hypothesis we kinetically characterized the selenocysteine containing selenophosphate synthetase from H. influenzae and compared its catalytic activity to that of the cysteine containing selenophosphate synthetase from E. coli. Our characterization revealed the Km values for the two substrates, selenide and ATP, were similar for both enzymes. However, the selenocysteine-containing enzyme did not exhibit the expected higher catalytic activity. Based on these results we suggest a role of selenocysteine in H. influenzae that is not catalytic.
Lacourciere
G M
GM
Laboratory of Biochemistry, National Heart, Lung, Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Stadtman
T C
TC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000201:enzymology*; D006193:Haemophilus influenzae / Q000201:enzymology*; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018036:Selenium Compounds / Q000378:metabolism; D017279:Selenocysteine; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D029721:Drosophila Proteins; D011994:Recombinant Proteins; D018036:Selenium Compounds; D017279:Selenocysteine; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C062762:selenophosphate synthetase; D003545:Cysteine