An engineered change in substrate specificity of ribulosebisphosphate carboxylase/oxygenase

The potential for altering the specificity of ribulosebisphosphate carboxylase/oxygenase toward gaseous substrates is explored through a modest perturbation of the active site microenvironment. Specifically, replacement of active site Glu-48 with carboxy-methylcysteine is achieved in a two-step process in which the catalytically incompetent Cys-48 mutant protein is first generated and then treated with iodoacetic acid. This regimen of concerted site-directed mutagenesis and chemical modification, effectively lengthening the glutamyl side chain by insertion of a sulfur atom between the beta- and gamma-methylene groups, results in a protein possessing 4-6% of wild-type carboxylase activity. Concomitantly, the engineered enzyme exhibits a specificity factor 5-fold lower than that of wild-type enzyme. This represents the first example of a major change in substrate specificity, albeit in favor of oxygenation, effected by structural alteration of an active site side chain.