SERINE-376 CONTRIBUTES TO THE BINDING OF SUBSTRATE BY RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE FROM ANACYSTIS-NIDULANS

Site-directed mutagenesis was used to change Ser376 in the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans to Cys, Thr, or Ala. When expressed in Escherichia coli and purified, the mutant enzymes exhibited carboxylase activities that were reduced by 99% or more with respect to the activity of the wild-type enzyme. The K(m) values for ribulose bisphosphate at pH 8.0, 30-degrees-C, were elevated from 46-mu-M for wild-type enzyme to 287, 978, and 81-mu-M for mutants in which Cys, Thr, or Ala, respectively, replaced Ser376. The Cys and Thr variants were almost devoid of oxygenase activity whereas the Ala variant had 16% as much oxygenase as wild-type enzyme, suggesting that this mutation had greatly elevated the oxygenase:carboxylase ratio.