Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase
Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 m s at 2C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with UC-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with UC-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence, two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. The reactive sulfhydryl groups are likely to be those generated by reduction of a disulfide during activation. 20 references, 3 figures, 2 tables.
- Research Organization:
- Oak Ridge National Lab., TN
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 6297749
- Journal Information:
- Arch. Biochem. Biophys.; (United States), Journal Name: Arch. Biochem. Biophys.; (United States) Vol. 236:2; ISSN ABBIA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ADP
ALKYLATION
AMINO ACIDS
ATP
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
COVALENCE
CYSTEINE
DATA
ENZYME ACTIVITY
ENZYMES
EXPERIMENTAL DATA
FOOD
INFORMATION
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
NUCLEOTIDES
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PLANTS
REACTION KINETICS
REAGENTS
RECEPTORS
SPINACH
THIOLS
TRACER TECHNIQUES
TRANSFERASES
VEGETABLES