Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification.
Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002219:Carbamates / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000235:genetics; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D009154:Mutation; D011487:Protein Conformation; D012247:Rhodospirillum rubrum / Q000201:enzymology / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000302:isolation & purification
D002219:Carbamates; D005971:Glutamates; D018698:Glutamic Acid; D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine