Catalytic properties of selenophosphate synthetases: Comparison of the selenocysteine-containing enzyme from Haemophtius influenzae with the corresponding cysteine-containing enzyme from Escherichia coli

摘要

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 res- idue 16 as carboxymethyl selenocysteine, which corresponded to tbe essential cysteine residue in the glycine-rich sequeuce 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 hypoth- esis we kinetically characterized the selenocysteine containing selenophosphate synthetase from H. influenzae and compared its catalytic activity to that of the cysteine containing seleno- phosphate synthetase from E. coli. Our characterization re- vealed the K_m values for the two substrates, selenide and ATP, were similar for both enzymes. However, the selenocysteine- containing enzyme did not exhibit the expected higher cata- lytic activity. Based on these results we suggest a role of selenocysteine in H. influenzae that is not catalytic.