Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.
The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme, S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparent Kd of 3-4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.
Porter
M A
MA
University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences 37831.
Potter
M D
MD
Hartman
F C
FC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism*; D000478:Alkylation; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004229:Dithiothreitol / Q000494:pharmacology; D004983:Ethanolamines / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D010449:Peptide Mapping; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology*; D011489:Protein Denaturation; D012274:Ribulosephosphates / Q000494:pharmacology
D000345:Affinity Labels; D004983:Ethanolamines; D012274:Ribulosephosphates; C031524:ribulose 5-phosphate; C020161:N-bromoacetylethanolamine phosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D003545:Cysteine; D004229:Dithiothreitol