Spinach leaf ribulose-5-phosphate kinase: examination of sulfhydryls by chemical modification and spin-labeling.
Methodology has been developed for complete or selective modification of the cysteinyl sulfhydryls of ribulose-5-phosphate (Ru5P) kinase. Using native enzyme, iodoacetate modifies four sulfhydryls with varying levels of completeness. The most reactive sulfhydryl in the native enzyme can be selectively titrated with iodoacetate; complete loss of activity occurs. Composition and N-terminal analyses of the peptide bearing this essential sulfhydryl indicate that the alkylated residue (Cys-16) is identical to the site modified by other modification reagents (M. A. Porter and F. C. Hartman (1986) Biochemistry 25, 7314-7318). In the presence of ATP, a nonessential sulfhydryl of the native enzyme is carboxymethylated. The peptide bearing this modified cysteine has been isolated and its composition and N-terminal sequence determined. Enzyme that is carboxymethylated in the presence of ATP retains activity and can be oxidatively inactivated in a reversible fashion. This suggests that the cysteine targeted by iodoacetate in the presence of ATP is not a residue that participates in regulation of enzyme activity. Using a spin-labeled analog of iodoacetate, both essential and nonessential cysteines have been selectively modified. ESR measurements suggest that the environment of these cysteines is not highly constrained. Modest effects on spin-label mobility are observed upon occupancy of Ru5P or ATP sites on the modified enzyme. These effects are dependent on the presence of divalent cations, suggesting that a binary enzyme-cation complex must form prior to productive enzyme-substrate interactions.
D000596:Amino Acids / Q000032:analysis; D002845:Chromatography / Q000379:methods; D003545:Cysteine / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D007461:Iodoacetates; D019807:Iodoacetic Acid; D010449:Peptide Mapping; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology*; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis*
D000596:Amino Acids; D007461:Iodoacetates; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D003545:Cysteine; D019807:Iodoacetic Acid