MUTATION OF ASPARAGINE-111 OF RUBISCO FROM RHODOSPIRILLUM-RUBRUM ALTERS THE CARBOXYLASE OXYGENASE SPECIFICITY

The conserved asparagine 111 of ribulose-1,5-bisphosphate carboxylase/oxygenase from the photosynthetic bacteria Rhodospirillum rubrum was identified as a candidate for a side-chain that might be involved in the carboxylase/oxygenase specificity. It was replaced by site-directed mutagenesis with aspartic acid, leucine, glutamine or glycine residues. The mutant enzymes exhibit a very low carboxylase activity compared with the wild-type enzyme. The values of Km(RuBp) and kcat for Asn111 → Gly, the most active mutant, are 420 μm and 0.034 s-1, compared with 13 μm and 3.0 s-1 for wild-type. The mutation of Asn111 → Gly causes a more than tenfold decrease in the CO2 O2 specificity factor, τ, τAsn111 → Gly = 0.56 and τwlld-type = 6.7. This is the first reported change in rubisco specificity by a single site-directed mutation alone and suggests a target for future protein engineering studies. © 1992.