Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis.
Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.
D000409:Alanine; D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D009154:Mutation; D010428:Pentosephosphates; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013402:Sugar Alcohols; D013403:Sugar Phosphates / Q000378:metabolism
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D010428:Pentosephosphates; D013402:Sugar Alcohols; D013403:Sugar Phosphates; D002245:Carbon Dioxide; C032687:2-carboxyarabinitol 1,5-biphosphate; D006639:Histidine; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium; D000409:Alanine