Intersubunit location of the active site of ribulose-bisphosphate carboxylase/oxygenase as determined by in vivo hybridization of site-directed mutants.

Abstract

Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50.5-kDa subunits with two substrate binding sites per molecule of dimer. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interactions, we have used a novel in vivo approach for producing heterodimers from catalytically inactive, site-directed mutants of the carboxylase. When the alleles encoding these mutant proteins are placed separately into compatible plasmids and coexpressed in the same Escherichia coli host, activity is observed at about 20% of the wild-type level. Analysis of the carboxylase purified from these cells reveals the presence of heterodimers of the two mutant proteins. This interallelic complementation demonstrates that domains from each of the subunits interact to form a shared active site.