Serine-376 contributes to the binding of substrate by ribulose-bisphosphate carboxylase/oxygenase from Anacystis nidulans

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Abstract
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 Km values for ribulose bisphosphate at pH 8.0, 30 degrees C, were elevated from 46 microM for wild-type enzyme to 287, 978, and 81 microM 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.

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