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 microM and 0.034 s-1, compared with 13 microM 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, tau, tau Asn111----Gly = 0.56 and tau wild-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.
Chène
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Department of Chemistry, University of Cambridge, U.K.
Day
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AG
Fersht
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AR
D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D012274:Ribulosephosphates / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012274:Ribulosephosphates; D001216:Asparagine; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium