Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16
- Univ. of Tennessee, Oak Ridge (United States)
- Oak Ridge National Lab., TN (United States)
The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys 16 is an active-site reside positioned at the nucleotide-binding domain. 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 K{sub d} of 3-4mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of ({sup 14}C)reagent per mole of enzyme subunit. Amino acid analysis of the ({sup 14}C)-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.
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 7261102
- Journal Information:
- Journal of Protein Chemistry; (United States), Journal Name: Journal of Protein Chemistry; (United States) Vol. 9:4; ISSN JPCHD; ISSN 0277-8033
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKYLATION
AMINO ACID SEQUENCE
AMINO ACIDS
ATP
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CARBON COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CYSTEINE
ENZYME ACTIVITY
ENZYMES
FOOD
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LABELLING
MAGNOLIOPHYTA
MAGNOLIOPSIDA
METABOLISM
METHYLATION
MOLECULAR STRUCTURE
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PLANTS
PROTEINS
REACTION KINETICS
REAGENTS
SPINACH
THIOLS
TRACER TECHNIQUES
TRANSFERASES
VEGETABLES